No abstract available.
Bone Marrow/metabolism/pathology ; DNA Mutational Analysis ; Gene Rearrangement, T-Lymphocyte ; Humans ; Immunophenotyping ; Infant ; Lymphohistiocytosis, Hemophagocytic/*diagnosis ; Male ; Membrane Proteins/chemistry/genetics ; Polymorphism, Single-Stranded Conformational ; Republic of Korea ; T-Lymphocytes/immunology/*metabolism

AAV-ITR single strand DNA mini vector (AAV-ITR ssDNA mini vector) is a novel gene expression vector based on AAV-ITR. We have shown efficient gene expression of AAV-ITR ssDNA mini vector in HEK 293T. Here, we studied the efficacy of gene expression of AAV-ITR ssDNA mini vector in vivo. We injected the skeletal muscle of ICR mice separately with equal molars of AAV-ITR ssDNA mini vector, ITR mutated AAV-ITR single strand DNA mini vector (AAV-ITRmm ssDNA mutant vector), AAV-ITR dsDNA and pUC57-minivector-GFP, combined with TurboFect. Florescence microscope analysis of skeletal muscle section shows that AAV-ITR ssDNA mini vector had higher expression efficiency and longer expression period. We extracted DNA from the muscle three months after injection and quantified three vectors by Real-time PCR. RT-PCR analysis shows that there were highest copy numbers of AAV-ITR ssDNA mini vector existing in muscle. Stable existing of AAV- TR ssDNA mini vector in muscle could be the molecular basis of long term gene expression of the vector. The results suggest that AAV-ITR ssDNA mini vector might be a promising vector for gene therapy.
Animals ; DNA, Single-Stranded ; genetics ; Dependovirus ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Mice ; Mice, Inbred ICR ; Muscle, Skeletal ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism* ; DNA ; DNA, Single-Stranded/genetics* ; In Vitro Techniques ; Ketamine/metabolism* ; Oligonucleotides ; SELEX Aptamer Technique/methods*

OBJECTIVE: To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2). METHODS: CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed. RESULTS: Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers. CONCLUSION C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.
Adolescent ; Adult ; Antigens, CD34 ; genetics ; immunology ; Antigens, Differentiation, Myelomonocytic ; genetics ; immunology ; Aptamers, Nucleotide ; genetics ; metabolism ; DNA, Single-Stranded ; genetics ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; immunology ; Male ; Middle Aged ; SELEX Aptamer Technique ; Sialic Acid Binding Ig-like Lectin 3 ; genetics ; immunology ; Young Adult

PARP is an important protein in DNA repair pathways especially the base excision repair (BER). BER is involved in DNA repair of single strand breaks (SSBs). If BER is impaired, inhibiting poly(ADP-ribose) polymerase (PARP), SSBs accumulate and become double stand breaks (DSBs). The cells with increasing number of DSBs become more dependent on other repair pathways, mainly the homologous recombination (HR) and the nonhomologous end joining. Patients with defective HR, like BRCA-deficient cell lines, are even more susceptible to impairment of the BER pathway. Inhibitors of PARP preferentially kill cancer cells in BRCA-mutation cancer cell lines over normal cells. Also, PARP inhibitors increase cytotoxicity by inhibiting repair in the presence of chemotherapies that induces SSBs. These two principles have been tested clinically. Over the last few years, excitement over this class of agents has escalated due to reported activity as single agent in BRCA1- or BRCA2-associated ovarian or breast cancers, and in combination with chemotherapy in triple negative breast cancer. This review covers the current results of clinical trials testing those two principles. It also evaluates future directions for the field of PARP inhibitor development.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Benzamides ; administration & dosage ; Benzimidazoles ; administration & dosage ; Breast Neoplasms ; drug therapy ; enzymology ; genetics ; DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; DNA End-Joining Repair ; DNA Repair ; Enzyme Inhibitors ; therapeutic use ; Female ; Genes, BRCA1 ; Genes, BRCA2 ; Homologous Recombination ; Humans ; Mutation ; Ovarian Neoplasms ; drug therapy ; enzymology ; genetics ; Phthalazines ; administration & dosage ; Piperazines ; administration & dosage ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library.

METHODSWith a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.

RESULTSAfter 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133.

CONCLUSIONWe have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.

AC133 Antigen ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; DNA, Single-Stranded ; Glycoproteins ; metabolism ; Humans ; Neoplastic Stem Cells ; metabolism ; Peptide Library ; Peptides ; metabolism ; Protein Binding ; Sequence Analysis, DNA

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals ; DNA Repair ; genetics ; DNA, Single-Stranded ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; genetics ; metabolism ; Loss of Heterozygosity ; genetics ; RecQ Helicases ; genetics ; metabolism

Numerous studies and clinical trials have demonstrated the efficacy of recombinant adeno-associated virus gene delivery vectors. However, prior to expression, it is necessary to convert the single-stranded DNA genome into double-stranded DNA, which hinders the efficiency of these vectors. We can entirely circumvent this step through the use of self-complementary recombinant adeno-associated virus vector (scrAAV). ScrAAV packages an inverted repeat genome that can fold into double-stranded DNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. By using scrAAV, we could increase expression efficiency and reduce immune response caused by vectors themselves. Therefore, it is a promising vector for gene therapy. So far, it has been used in the treatment of hepatic diseases, central nervous system diseases, and eye diseases. It has also been used in the modifications of stem cells and as vectors for siRNA/miRNA and ribozymes. In this review, we focused on the preparation, expression and location of scrAAV both in vitro and in vivo. We mainly introduced the recent progress of scrAAV based therapy of Hemophilia B, in order to elucidate the potential and prospects of scrAAV in gene therapy.
Animals ; Base Sequence ; DNA ; genetics ; DNA, Complementary ; genetics ; DNA, Single-Stranded ; genetics ; Dependovirus ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Therapy ; methods ; trends ; Genetic Vectors ; genetics ; Hemophilia B ; therapy ; Humans ; Molecular Sequence Data

OBJECTIVETo investigate the clinicopathological and biological features of micrometastasis in early gastric cancers.

METHODSEleven cases of early gastric cancer with micrometastasis (micrometastatic, MM group) and 46 cases of early gastric cancer with lymph node metastasis (control group) were included in the study. Immunochemical staining of ssDNA, bcl-2, p53, E-cadherin, Ki-67, CD34 was performed. The superficial lesions, invasive fronts and lymph nodes were examined in both groups.

RESULTSPositive rate of ssDNA at the superficial lesions in MM group was higher than that in control group. In MM group the positive rate of ssDNA in micrometastasis was higher than that at invasive fronts and in lymph nodes. Positive rate of bcl-2 at the superficial lesions in micrometastasis was higher than that at invasive fronts and lymph nodes. Positive rate of c-myc at the superficial lesions in MM group was higher than that in control group. Positive rate of E-cadherin and the percentage of microvascular areas at the lymph nodes in MM group was lower than those in control group. Proliferative ability of cancer cells at superficial lesions and lymph nodes in MM group was lower than those in control group. Lymph nodes <3 mm in micrometastasis accounted for 27.3%.

CONCLUSIONThe pathological and biological features of micrometastasis in early gastric cancer show low positive rate of ssDNA, E-cadherin, Ki-67 and low percentage of microvascular areas at the lymph nodes.

Adenocarcinoma ; metabolism ; pathology ; Antigens, CD34 ; analysis ; Biomarkers, Tumor ; analysis ; Cadherins ; analysis ; DNA, Single-Stranded ; analysis ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; analysis ; Lymphatic Metastasis ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Stomach Neoplasms ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo detect the mutations of BRCA1 and BRCA2 in sporadic breast cancer and study the relationship between BRCA1 and BRCA2 mutations and breast cancer.

METHODSBreast cancer tissues of 144 patients and breast tissues of 30 cases of healthy people who were treated from December 2000 to September 2005 were studied. DNA was extracted by the phenol-chloroform method. Fragments of exon 2, exon 3, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23 and exon 24 in the BRCA1 gene and exon 10 and exon 14 in the BRCA2 gene were amplified by polymerase chain reaction. Mutation screening was performed by single-strand conformation polymorphism analysis and alterations were confirmed by DNA sequencing.

RESULTSA total of 20 single nucleotide changes in BRCA1 were detected in the 144 cases of breast cancer patients. The total mutation rate was 13.9% and missense mutation rate was 11.1%. No mutation was detected in the BRCA2 and controls.

CONCLUSIONSMutations in BRCA1 may play an important role in evaluation of sick risk, earlier diagnosis and gene therapy of breast cancer in southern Chinese populations.

Adult ; Aged ; Apoptosis Regulatory Proteins ; BRCA1 Protein ; genetics ; metabolism ; BRCA2 Protein ; genetics ; metabolism ; Base Sequence ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; DNA Mutational Analysis ; Female ; Humans ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational