This work was directed at obtaining a better gene carrier to improve the effects of gene delivery. Neutral liposomes made from cholesterol, lecithin and DOPE by reverse evaporation technique were used for encapsulating DNA-HAP complex which was made from DNA and optimized HAP. The sizes of complexes and the efficiency of encapsulation were detected. The efficiency of transfection into Hela cells was shown by observation of X-gal staining and measurement of transfection efficience. The average size of complexes was 643nm, the average encapsulating efficiency of DNA in microspheres reached 11.67%. These Lipid-Hydroxyapatite-DNA complex (LHD) could be transfected into mammalian cells. The Lipid-Hydroxyapatite-DNA complex prepared by reverse evaporation technique could be applied availably in DNA delivery system, and it gave another thinking to increase the gene transfection of non- viral genetic vector.
DNA ; administration & dosage ; Durapatite ; administration & dosage ; Genetic Therapy ; Lipids ; administration & dosage ; Transfection ; methods

Maternal nutrition during pregnancy plays a vital role in the health of the offspring. Methyl donor nutrients, including folate, vitamin B, choline, betaine, and methionine, directly affect DNA methylation and are closely associated with the health of the offspring. As an important part of epigenetics, DNA methylation plays an important role in the maintenance of normal cellular function, gene expression regulation, and embryonic development. Recent studies have shown that maternal nutrition may have a long-lasting effect on the health of the offspring via the changes in genomic DNA and/or methylation level in the promoter region in specific genes. Therefore, this review article focuses on the effect of maternal intake of methyl donor nutrients during pregnancy on DNA methylation, in order to explore the effect of the changed methylation status on the health of the offspring at the molecular level.
Betaine ; administration & dosage ; Choline ; administration & dosage ; DNA Methylation ; Female ; Folic Acid ; administration & dosage ; Humans ; Maternal Nutritional Physiological Phenomena ; Methionine ; administration & dosage ; Pregnancy ; Vitamin B 12 ; administration & dosage

This work reports the properties of dextran-spermine polycation (DSP) as a gene vector and its gene transfection efficiency in vitro. Oxidized dextran was reacted by reductive amination with spermine to obtain DSP, the resulted polycation was then incubated with plasmid pEGFP to form polyplexes by electrostatic interactions. DSP formed stable polyplexes when the weight ratio (DSP/DNA) varied from 4 : 1 to 20 : 1. The particle size and zeta potential of polyplexes were in the range of 162.6 - 187.9 nm and increased from + 8.45 mV to + 39.6 mV, respectively. DSP could effectively protect condensed DNA from DNase I degradation, and it showed strong buffering capacity in a certain pH range. The highest yields of transfection efficiency were found to be as high as Lipofectamine 2000 when the polyplexes were transfected to SMMC-7721 and BHK-21 cells at the weight ratio of 8 : 1. This research indicates that dextran-spermine polycation is a highly active gene vector in vitro.
Animals ; Cell Survival ; Cricetinae ; DNA ; administration & dosage ; Dextrans ; administration & dosage ; Genetic Vectors ; Spermine ; administration & dosage ; Transfection ; methods

Animals ; Cations ; chemistry ; Chitin ; administration & dosage ; analogs & derivatives ; chemistry ; Chitosan ; DNA ; administration & dosage ; Gene Transfer Techniques ; Genetic Therapy ; Liposomes ; administration & dosage ; chemistry ; Polylysine ; administration & dosage ; chemistry

The aim was to investigate the effect of particle size on transfection efficiency of chitosan (CS)-based nanoparticles. Nanoparticles were synthesized through complex coacervation CS with plasmid DNA (pDNA). Three kinds of pDNA/CS nanoparticles with different sizes (250, 580 and 1300 nm) were prepared by altering the adding rate and the vortexing time. The particle size, zeta potential and the stability in cultural medium were evaluated by zetasizer. The association efficiency was determined by spectrofluorophotometer. The combination of chitosan with pDNA as well as the ability to protect pDNA from nuclease degradation was analyzed by gel electrophoresis. The transfection efficiency of pDNA/CS nanoparticles in HEK293 cells was investigated by flow cytometry. Using CS grafted fluorescein isothiocyanate as a fluorescent marker, the adsorption features of the nanoparticles were visualized by fluorescence microscopy and the cellular uptake percent was quantitated by flow cytometry. The internalization process of the nanoparticles was visualized by confocal laser scanning microscopy (CLSM) using nanoparticles of the size of 250 nm. Results showed that the three kinds of pDNA/CS nanoparticles had no differences in zeta potential, association efficiency, protection ability, stability and transfection efficiency in HEK293. The nanoparticles were all adsorbed on cell surface in the form of aggregates, and similar cellular uptake percent as well as quantities were observed 4 h post-incubation with HeLa cells. CLSM images showed that the aggregates below 2 microm could be internalized by endocytosis. These results suggest that the transfection efficiency of pDNA/CS nanoparticles does not depend on particle size in the range from 250 nm to 1300 nm.
Chitosan ; administration & dosage ; chemistry ; metabolism ; DNA ; administration & dosage ; Endocytosis ; Genetic Vectors ; HeLa Cells ; Humans ; Nanoparticles ; Particle Size ; Plasmids ; Transfection

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patient's ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.
*Sequence Analysis, DNA ; RNA, Ribosomal, 18S/genetics ; Polymorphism, Restriction Fragment Length ; Parasitic Sensitivity Tests ; Myopia/therapy ; Itraconazole/administration & dosage ; Humans ; Female ; Disinfectants/administration & dosage ; DNA, Ribosomal/analysis ; DNA, Protozoan/analysis ; DNA, Mitochondrial/analysis ; Contact Lenses/*adverse effects ; Chlorhexidine/administration & dosage ; Biguanides/administration & dosage ; Astigmatism/therapy ; Antiprotozoal Agents/administration & dosage ; Animals ; Adolescent ; Acanthamoeba Keratitis/drug therapy/*parasitology ; Acanthamoeba/classification/*genetics/*isolation & purification

OBJECTIVETo investigate the inhalation of sulfur dioxide (SO(2)) on the DNA damage of brain cells in mammalian animals.

METHODSingle cell microgel electrophoresis technique (comet test) was used to test the DNA damage of brain cells.

RESULTS(1) After the exposure to SO(2) at 0, 7, 14, 28 mg/m(3), the tail lengths of nuclear DNA in brain cells from male mice were 8.02, 23.14, 46.43 and 53.49 micro m respectively; and that from female mice were 7.23, 12.43, 20.39 and 54.83 micro m respectively. The results showed that: (1) SO(2) inhalation caused damage on DNA of brain cells in a dose-dependent manner; (2) Even under lower concentration of SO(2) as 7 mg SO(2)/m(3), the damage on DNA of brain cells was also reached to 98.8%. It implied that the brain cells of mammalian animals were very sensitive to SO(2) inhalation; (3) The DNA damage of brain cells from male mice is more serious than that from female mice. The reasons remain to be further studied.

CONCLUSIONSSO(2) pollution even at lower concentrations also had a potential risk to the genetic material DNA of brain cells from mammalian animals. The results of our study might explain the recently published epidemiological studies that the workers exposed to SO(2) or SO(2) derivatives had suffered an increase of mortality from brain cancer.

Administration, Inhalation ; Animals ; Brain ; drug effects ; metabolism ; DNA ; DNA Damage ; Dose-Response Relationship, Drug ; Female ; Male ; Mice ; Sulfur Dioxide ; toxicity

Mushrooms have become a largely untapped source of powerful new pharmaceutical products that poses anti-inflammatory, and antimutagenic, and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protecting cellular components against free radical. The aim of this study was to investigate the protective effect of chaga mushroom against diabetes, via the mitigation of oxidative stress and reduction of blood glucose, in streptozotocininduced diabetic rats. Rats were rendered diabetic by intravenous administration of STZ through tail at a dose of 50 mg/kg. Animals were allocated into four groups with 8 rats each. The control and diabetic control group were fed withstandard rat feed. The other diabeic groups, the low chaga extract group and the high chaga extract group were fed ad libitum using 0.5 g/kg and 5 g/kg of chaga mushroom extract, respectively, for 4 weeks. The blood glucose levels in the two chaga extract groups showed a tendency to decrease but did not reach statistical significance after the supplementation. Leukocyte DNA damage, expressed as tail length, was found to be significantly lower in the high chaga extract group than in the diabetic control group (p > 0.05). Plasma level of total radical-trapping antioxidant potential (TRAP) was tend to be higher in the high chaga extract group compared with the diabetic control group. Erythrocyte antioxidant enzyme activities of two groups did not differ. Although we did not obtain beneficial effect on lowering blood glucose levels in the STZ-induced diabetic rats, this results suggest that the chaga mushroom extracts may initially act on protecting endogenous DNA damage in the short-term experiment.
Administration, Intravenous ; Agaricales ; Animals ; Antioxidants ; Blood Glucose ; DNA ; DNA Damage ; Erythrocytes ; Leukocytes ; Oxidative Stress ; Pharmaceutical Preparations ; Plasma ; Rats ; Streptozocin ; Tail

OBJECTIVESTo study antibody response to a hepatitis B DNA vaccine by formulation with aluminum phosphate in mice.

METHODSAn eukaryotic expression plasmid inserted HBsAg gene (pcDNA3.1-S) was constructed by cloning technique and the accuracy of the construct was confirmed by restriction enzyme digestion and DNA sequencing, then hepatitis B DNA vaccine formulations were prepared by mixing pcDNA3.1-S with various concentration of aluminum phosphate in 0.9% NaCl. HBsAg expressions were assayed by ELISA in vivo five days after intramuscular injection of pcDNA3.1-S with or without aluminum phosphate. And serum samples were obtained from individual immunized or control mice 6 weeks post injection. Then anti-HBs were assayed in mice sera by ELISA.

RESULTSFive days after intramuscular immunization, the levels of HBsAg expression of groups with aluminum phosphate showed no difference from those of control group in tibialis arterials muscles. In sera, HBsAg could not be detectable in all groups. Intramuscular immunization of BABL/C mice with pcDNA3.1-S mixed aluminum phosphate (0microg, 1microg, 10microg, 50microg, 100microg) 6 weeks later, the P/N values of anti-HBs in sera were 11.54+/-5.60, 11.00+/-6.62, 20.30+/-10.20, 49.18+/-24.40 and 48.68+/-27.78, respectively. It showed that pcDNA3.1-S mixing with aluminum phosphate could increase anti-HBs titers in mice.

CONCLUSIONNo increase of HBsAg expression was observed by mixing plasmid pcDNA3.1-S with various concentration of aluminum phosphate in vivo. But Intramuscular immunization of BALB/C mice with pcDNA3.1-S mixing aluminum phosphate adjuvant can increase anti -HBs titers. It seemed that aluminum phosphate would be valuable for further investigation as a potential adjuvant of hepatitis B DNA vaccines.

Adjuvants, Immunologic ; administration & dosage ; Aluminum Compounds ; administration & dosage ; Animals ; Female ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Mice ; Mice, Inbred BALB C ; Phosphates ; administration & dosage ; Vaccines, DNA ; administration & dosage ; immunology

OBJECTIVETo investigate the effect of a phosphorothioate antisense oligodeoxynucleotide "ASOND" combined with cis-Diamminedichloroplatinum (DDP), 5-fluorouracil (5-FU) and adriamycin (ADM) respectively on inhibiting the proliferation of HepG2 cells.

METHODSA phosphorothioate antisense oligodeoxynucleotide (5'-ACTCACTCAGG CCTCAGACT-3') targeted to human telomerase reverse transcriptase (hTERT) mRNA, which named cantide, was synthesized. ASODN was transfected into HepG2 by lipofectin. And cell growth activity was evaluated by MTT assay. SAS software and Jin Zhengjun Method were used to evaluate the interaction of ASODN and these chemotherapeutic drugs.

RESULTSCombination treatments with 0.1micromol/L ASODN reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29microg/ml to 0.25, 1.52 and 0.12microg/ml respectively. The inhibitory ability of combination treatments on HepG2 cells was higher than that of these drugs alone (F=66.92, 25.96, 8.56, P<0.001). And synergism (Q>or=1.15) was observed at the lower concentration of DDP (

CONCLUSIONASODN may enhance therapeutic effectiveness of chemotherapeutic drugs in human hepatocellular carcinoma cells.

Antineoplastic Agents ; administration & dosage ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; DNA-Binding Proteins ; Doxorubicin ; administration & dosage ; Drug Synergism ; Fluorouracil ; administration & dosage ; Humans ; Liver Neoplasms ; drug therapy ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; Telomerase ; antagonists & inhibitors ; genetics