In vitro gene delivery to HepG2 cells using galactosylated 6-amino-6-deoxychitosan as a DNA carrier

A chitosan derivative, 6-amino-6-deoxy chitosan (6ACT), was galactosylated and was investigated as a gene carrier. A series of galactose-modified 6ACT (Gal-6ACT) with degrees of substitution (d.s.) ranging from 3% to 50% per pyranose were prepared by reductive alkylation with lactose. DNA retardation assays showed that the electrostatic interaction between Gal-6ACT and plasmid DNA was not changed by galactose modification up to 50% per pyranose of 6ACT. Gal-6ACT with a d.s. of 38% was bound to galactose-recognizing lectin, RCA120. A significant increase in transfection efficiency for HepG2 cells was observed at degree of substitutions ranging from 18% to 50% and at N/P values ranging from 1.5 to 2.5. Under optimum conditions, Gal-6ACT showed about 10 times higher efficiency than 6ACT. However, a slight uptake by the galactose receptors on hepatocytes was observed by flow cytometric analysis. Moreover, Gal-6ACT with a d.s. of 38% mediated efficient gene transfer into both A549 and HeLa cells lacking the galactose receptor. These results suggest that the enhancement of transfection efficiency of Gal-6ACT was not due to the increase of receptor-mediated cellular uptake. In addition, the enhanced gene transfer efficiency was not specific to the galactose modification because the efficiency of glucose-modified 6ACT for HepG2 cells was similar as that of Gal-6ACT.     

Synthesis and characterization of dexamethasone‐conjugated linear polyethylenimine as a gene carrier

Linear polyethylenimine (25 kDa, LPEI25k) has been shown to be an effective non‐viral gene carrier with higher transfection and lower toxicity than branched polyethylenimine (BPEI) of comparable molecular weight. In this study, dexamethasone was conjugated to LPEI25k to improve the efficiency of gene delivery. Dexamethasone is a synthetic glucocorticoid receptor ligand. Dexamethasone‐conjugated LPEI25k (LPEI–Dexa) was evaluated as a gene carrier in various cells. Gel retardation assays showed that LPEI–Dexa completely retarded plasmid DNA (pDNA) at a 0.75:1 weight ratio (LPEI/pDNA). LPEI–Dexa had the highest transfection efficiency at a 2:1 weight ratio (LPEI–Dexa/DNA). At this ratio, the size of the LPEI–Dexa/pDNA complex was approximately 125 nm and the zeta potential was 35 mV. LPEI–Dexa had higher transfection efficiency than LPEI and Lipofectamine 2000. In addition, the cytotoxicity of LPEI–Dexa was much lower than that of BPEI (25 kDa, BPEI25k). In conclusion, LPEI–Dexa has a high transfection efficiency and low toxicity and can therefore be used for non‐viral gene delivery. J. Cell. Biochem. 110: 743–751, 2010. © 2010 Wiley‐Liss, Inc.     

Preparation and characterization of N-(2-carboxybenzyl)chitosan as a potential pH-sensitive hydrogel for drug delivery

A novel water-soluble chitosan derivative [N-(2-carboxybenzyl)chitosan, CBCS] was synthesized. The chemical structure of CBCS was characterized by FTIR, (1)H NMR and UV spectroscopies. The degree of substitution (DS) of N-2-carboxybenzyl was determined by colloid titration. In different pH buffer solutions, the swelling characteristics of hydrogels based on CBCS (CBCSG) prepared by crosslinking with glutaraldehyde have been studied. Results showed that the swelling ratio (SR) of CBCSG decreased with an increase of the amount of glutaraldehyde, and that CBCSG swelled more significantly in alkaline solution than in acidic medium, showing the lowest SR at pH5.0. The SR of CBCSG increased with the raising of the DS of the N-2-carboxybenzyl group in alkaline solution, but no significant change was observed in an acidic environment. CBCSG showed swelling reversibility when alternately soaked in pH1.0 and 7.4 buffer solutions. Release profiles of fluorouracil (5-FU), a poorly water-soluble drug, from CBCSG were studied under both simulated gastric and intestinal pHconditions. The release was much quicker in pH7.4 buffer than in pH1.0 solution. Results indicated that CBCS could be a potential pH-sensitive carrier for colon-specific drug delivery system.     

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.     

Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies

Polyethyleneimine (PEI) has been used previously as a nonviral DNA transfer vector. In this article, we demonstrate its use as a vehicle for transmembrane delivery of proteins in cell culture conditions. Linking proteins to PEI required no other treatment beyond mixing them with PEI. The bond between PEI and protein combined at optimal ratios was maintained in electrophoresis, even in the presence of 2.5% sodium dodecyl sulfate (SDS). The optimal time for delivery of proteins was determined to be 24 h. We have successfully delivered an Alexa 488-labeled avidin protein into human glioblastoma cells. A functional antibody against the nuclear protein lamin was delivered into human fibroblasts and reacted with lamin inside live cells. PEI-based delivery of antibodies and fluorescently labeled proteins can be used for fluorescent detection, tracking, and evaluation of cellular protein function in vivo.     

重组人肿瘤坏死因子α和白细胞介素6融合基因的构建和表达

本文利用ＰＣＲ技术和基因定位突变技术,将编码人肿瘤坏死因子α（ｈＴＮＦα）和白细胞介素６（ｈＩＬ－６）成熟肽的基因通过中间接头连接成编码单一蛋白的基因,构建了融合蛋白表达载体ｐＢＩＴ,并在大肠杆菌中得到了表达。ＳＤＳ－ＰＡＧＥ的电泳胶薄层扫描显示,融合蛋白的表达量是菌体总蛋白量的２０％,其分子量约为３７ｋＤ。活性检测证实,融合蛋白既有ＴＮＦ活性,又有ＩＬ－６活性。     

Design and evaluation of proniosomes as a carrier for ocular delivery of lomefloxacin HCl

The current investigation aims to develop and evaluate novel ocular proniosomal gels of lomefloxacin HCl (LXN); in order to improve its ocular bioavailability for the management of bacterial conjunctivitis. Proniosomes were prepared using different types of nonionic surfactants solely and as mixtures with Span 60. The formed gels were characterized for entrapment efficiency, vesicle size, and in vitro drug release. Only Span 60 was able to form stable LXN-proniosomal gel when used individually while the other surfactants formed gels only in combination with Span 60 at different ratios. The optimum proniosomal gel; P-LXN 7 (Span 60:Tween 60, 9:1) appeared as spherical shaped vesicles having high entrapment efficiency (>80%), appropriate vesicle size (187?nm) as well as controlled drug release over 12?h. Differential scanning calorimetry confirmed the amorphous nature of LXN within the vesicles. Stability study did not show any significant changes in entrapment efficiency or vesicle size after storage for 3 months at 4?°C. P-LXN 7 was found to be safe and suitable for ocular delivery as proven by the irritancy test. The antibacterial activity of P-LXN 7 evaluated using the susceptibility test and topical therapy of induced ocular conjunctivitis confirmed the enhanced antibacterial therapeutic efficacy of the LXN-proniosomal gel compared to the commercially available LXN eye drops.     

小菜蛾U6 snRNA基因的克隆及作为miRNA定量表达分析内参基因的评价

【目的】克隆小菜蛾Plutella xylostella (L.)小分子非编码RNA U6的cDNA序列,并评价其是否适合作为定量检测小菜蛾microRNA (miRNA)表达量的内参基因。【方法】本研究采用RT-PCR 克隆获得了小菜蛾4龄幼虫核小RNA(small nuclear RNA, snRNA) U6的cDNA序列,并用定量PCR法检测了U6及8种miRNAs在小菜蛾不同发育阶段及不同杀虫药剂处理后的表达稳定性。【结果】小菜蛾U6的cDNA序列全长 94 bp,与其他昆虫U6的核苷酸序列一致性达98.9%。用geNorm和RefFinder软件分析荧光定量PCR结果表明,U6在小菜蛾卵、1-4龄幼虫、蛹和成虫7个不同发育阶段表达稳定;用马拉硫磷、毒死蜱、辛硫磷、灭多威、呋喃虫酰肼、高效氯氰菊酯、氯虫苯甲酰胺、溴虫腈和Bt 9种不同作用机理的杀虫药剂处理3龄末幼虫48 h,对U6的表达水平无显著影响。【结论】小菜蛾U6表达水平不受不同发育阶段和不同杀虫药剂处理的影响,符合作为内参基因的基本特点,可作为定量PCR法评价小菜蛾miRNA或其他非编码小分子RNA表达水平的内参基因。研究结果为小菜蛾miRNA表达水平的准确定量奠定了基础。     

An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.     

Expression, purification and characterization of TAT-high mobility group box-1A peptide as a carrier of nucleic acids

High mobility group box 1 (HMGB1) is an abundant nuclear protein that binds to double-stranded DNA. HMGB1 is composed of high mobility (HMG) box A, box B, and C-terminal acidic regions. In this study, a recombinant TAT linked HMGB1 box A (rTAT-HMGB1A) peptide was expressed, purified, and characterized as a carrier of nucleic acids. The HMGB1A cDNA was amplified by PCR, and cloned into the pET21a expression vector with the TAT domain located at the N-terminus. The rTAT-HMGB1A peptide was overexpressed and purified using Nickel affinity chromatography. A recombinant HMGB1A (rHMGB1A) peptide without the TAT domain was also overexpressed and purified as a control. In gel retardation assays, both the rHMGB1A and rTAT-HMGB1A peptides formed complexes with DNA equally well. However, transfection assays showed that the rTAT-HMGB1A peptide had a higher gene transfer efficiency than rHMGB1A. Finally, rTAT-HMGB1A had no cytotoxicity to HEK 293 cells suggesting that rTAT-HMGB1A may be useful as a non-toxic gene delivery carrier.     

Comparative molecular characterization of gene segment 11-derived NSP6 from lamb rotavirus LLR strain used as a human vaccine in China

Sequence-length polymorphism is known for rotavirus genetic segment 11 (encodes non-structural protein, NSP6). With the exception of 11 strains that have the coding potential for a 98-residue NSP6, majority of the strains have the potential for a 92-residue NSP6. In nine strains, the coding potential for this protein is even shorter. This report focuses on the NSP6 gene nucleotide sequence of Lanzhou Lamb Rotavirus (LLR) strain and its comparative molecular characterization. The LLR strain is a G10 P12 type, which is in use as a licensed human vaccine in China. The LLR NSP6 was compared with 56 other rotaviral NSP6 sequences including a rhesus strain (RRV) available in the database. Analyses indicate that while RRV-NSP6 belongs to the majority (92-residue) group, the LLR NSP6 belongs to the 98-residue group. When the rotavirus NSP6 protein was expressed in cells as GFP fusion protein from human, simian and the LLR strains, they all demonstrated punctate cytoplasmic distribution and, contrary to the computer-aided prediction, the NSP6 did not undergo phosphorylation, which in itself is a novel observation for the rotavirus NSP6.     

High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator

Efficiency and reproducibility of gene electrotransfer depend on the electrical specifications provided by the pulse generator, such as pulse duration, pulse number, pulse frequency, pulse combination, and current intensity. Here, we describe the performances of GET42, a pulse generator specifically designed for gene electrotransfer into skeletal muscle. Expression of beta-galactosidase in the Tibialis anterior muscle of Sprague-Dawley male rats was increased 250-fold by GET42 compared to DNA injection alone. Combination of high and low current intensity pulses further increased transfection efficiency (400-fold compared to DNA injection without electrotransfer). Varying degrees of muscle necrosis were observed after gene electrotransfer. Nevertheless, muscle necrosis was dramatically reduced after optimization of cumulated pulse duration without significant reduction in transfection efficiency. Physiological applicability was illustrated by the analysis of cytochrome c promoter transactivation. In conclusion, GET42 has proven to be a reliable and efficient pulse generator for gene electrotransfer experiments, and provides a powerful mean to study in vivo the regulation of gene expression.     

同一滴度不同方法转染AAV9-SERCA2a基因对大鼠的有效性和毒副作用评价

目的研究同一滴度不同心脏基因转染方法转染携带肌浆网钙ATP酶2a(sarcoplasmic reticulumCa2+ATPase 2a,SERCA2a)基因的9型腺相关病毒(adeno-associated virus serotype 9,AAV9)即AAV9-SERCA2a对SD大鼠心肌的有效性及对机体毒副作用的影响。方法体外成功构建AAV9-SERCA2a-EGFP病毒载体系统,90只雄性SD大鼠随机分为尾静脉注射(tail vein injection,TVI)组、心肌注射(intramyocardial injection,IMI)组、心包腔注射(intrapericardial injection,IPI)组,采用相应方法在体分别转染滴度1×1011vg/mL AAV9-SERCA2a-EGFP、AAV9-EGFP、NS各200μL。转染后30 d,荧光显微镜下观察心肝肾组织绿色荧光表达情况,Western blot检测SER-CA2a基因在大鼠各组织的相对表达量,体表12导联心电图检测心律失常发生率,HE染色观察病理学变化,心脏彩色多普勒超声评价心功能,血液生化学指标检测肝肾功能。结果三组左心室可见大量绿色荧光表达,IMI组荧光局限在注射点,三组肝肾组织均可见微弱荧光;三组心肌SERCA2a蛋白相对表达量显著高于肝肾(P<0.01),TVI组和IMI组心肌SERCA2a蛋白相对表达量明显高于IPI组(P<0.01);TVI组和IPI组心电图正常,IMI组2只大鼠发生室性早搏;三组转染AAV9-EGFP、AAV9-SERCA2a-EGFP与转染NS相比,心功能、组织病理学、血液生化学指标差异均无显著性(P>0.05)。结论三种方法转染滴度1×1011vg/mL AAV9-SERCA2a基因,TVI法和IMI法在心肌的有效性优于IPI法,TVI法和IPI法对机体的毒副作用小于IMI法,综合三种方法的有效性和毒副作用评价,TVI法优于IMI法和IPI法。     

Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes the subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.     

Androgenic and anti-androgenic effects of progesterone derivatives with different halogens as substituents at the C-6 position.

The pharmacological activities of four pregnane derivatives: 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dio ne (7), 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dio ne (8), 17alpha-acetoxy-6-bromo-16beta-methylpregna-4,6-diene- 3,20-dione (10), and 17alpha-acetoxy-6-chloro-16beta-methylpregna-4,6-diene -3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 microg, stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11 involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing the electronegativity of the halogen at C-6 (compound 10), 5alpha-reductase is inhibited in both tissues and at different pHs. On the other hand, when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.