Erythromycin versus neomycin in the treatment of hepatic encephalopathy in cirrhosis: A randomized double-blind study

Background: Hepatic encephalopathy (HE) is a severe complication in patients with hepatic cirrhosis, which causes numerous hospital admissions and deaths. Antibiotics are the best options in HE treatment, but head-to-head comparisons between these drugs are scarce. Erythromycin combines the antimicrobial effect and prokinetic properties in the same drug, but it has never been used in HE treatment. Our aim was to evaluate the efficacy of erythromycin as an HE treatment.Methods: We achieved a randomized controlled trial of adult patients with HE and hepatic cirrhosis admitted in our hospital. After randomization, the subjects received either erythromycin 250 mg or neomycin 1 g orally QID until hospital discharge or prescription of another antibiotic. All subjects were blindly evaluated every day towards quantifying clinical, neuropsychometric, hepatic and renal exams. Statistical analysis was employed to compare the groups and correlate the variables with hospitalization duration.Results: 30 patients were evaluated (15 treated with each drug). At hospital admission, the groups were homogeneous, but the erythromycin group subjects achieved a shorter hospitalization stay (p = 0.032) and a more expressive reduction in alanine aminotranspherase levels (p = 0.026). Hospitalization duration was positively correlated with C reactive protein levels measured previous to (p = 0.015) and after treatment (p = 0.01).Conclusions: In the sample evaluated erythromycin was associated with significant reductions in hospital stay and in alanine aminotranspherase values. Hospitalization time was positive correlated with C reactive protein levels measured before and after the treatments. © 2013 Romeiro et al.; licensee BioMed Central Ltd.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Coincident sequence-specific RNA degradation of linked transgenes in the plant genome

The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neo(R) (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacE gene codes for β-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

Antibiotic sensitivity of Staphylococcus aureus strains isolated from fishery products

During the course of an investigation on the incidence of Staphylococcus aureus contamination in frozen crab meat, frozen prawns and dried fishes from Cochin, 116 strains of S. aureus were isolated. The sensitivity of the isolated strains towards nine antibiotics showed that all the S. aureus strains were sensitive to kanamycin and streptomycin (100%). Sensitivity to other antibiotics like chloramphenicol, polymyxin-B, erythromycin, tetracycline, neomycin, penicillin and ampicillin were shown by 98.28, 93.10, 87.93, 68.10, 67.24, 56.90 and 55.17 percent of the isolates respectively.

Antibiotic sensitivity of Vibrio parahaemolyticus strains

The strains of Vibrio parahaemolyticus isolated from water, sediment, plankton, fish and prawn of Cochin backwater were tested for sensitivity to ten antibiotics namely, ampicillin, chloramphenicol, gentamycin, kanamycin, neomycin, oxytetracycline, penicillin, polymyxin-B, streptomycin and sulphadiazine. Of the 120 isolates tested, 96.7 and 93.3% were sensitive to gentamycin and chloramphenicol respectively. No strain was sensitive to penicillin and only 5% were sensitive to kanamycin. Isolates from fish and prawn showed higher resistance to ampicillin and none of them was sensitive to kanamycin. Multiple resistant V. parahaemolyticus strains were more in prawn than in other samples.

Comparison of media and methods for the detection and enumeration of Clostridium perfringens in seafoods

Three direct plating methods and two most probable number (MPN) procedures were compared for the enumeration of Clostridium perfringens in seafoods the sulfitecycloserine (SC) agar, sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite- neomycin (TSN) agar, LS medium MPN procedure and iron milk MPN procedure. Isolates were confirmed as C. perfringens. The two MPN procedures compared very well with the three plating media tested with stock culture of C. perfringens from our laboratory collection and the reference strain NCIB 6125. But in fish samples, the two liquid media were found to be more sensitive and hence the MPN procedure using LS medium for the detection of C. perfringens in seafoods is suggested.

Antibiotic resistance of Staphylococcus aureus strains isolated from fish processing factory workers

One hundred and twenty two strains of Staphylococcus aureus isolated from throats and palms of 39 workers from 6 fish processing factories situated in and around Cochin were tested for their sensitivity to nine commonly used antibiotics-ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, penicillin, polymyxin-B, streptomycin and tetracycline. Highest percentage of resistance was observed towards ampicillin followed by penicillin i.e. 64.75% and 59.84%. Resistance towards other antibiotics like tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin were shown by 22.95, 16.39, 7.38, 5.74, 3.28 and 1.64% of the isolates respectively.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B-A transition in solution

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nano-device applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40 degrees C by SWNTs but no change for AT-DNA. SWNTs can induce B-A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B-A transition in solution and they bind to the DNA major groove with GC preference.

Caracterização da estrutura populacional de isolados de Escherichia coli provenientes de suiniculturas de produção intensiva e extensiva de Portugal

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Retroviral-mediated gene transfer of the human erythropoietin gene into a factor-dependent cell line, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.