scholarly journals rAAV Vectors as Safe and Efficient Tools for the Stable Delivery of Genes to Primary Human Chondrosarcoma CellsIn VitroandIn Situ

Sarcoma ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Henning Madry ◽  
Jagadeesh K. Venkatesan ◽  
Gertrud Schmitt ◽  
Sarah Schetting ◽  
Myriam Ekici ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Safe Delivery ◽  
E Coli ◽  
Human Chondrosarcoma ◽  
Gene Vectors ◽  
Transfer Strategies ◽  
Transfer Method ◽  
Metabolic Activities

Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV) vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cellsin vitroandin situwith different reporter genes (E. coli lacZ, fireflyluc, Discosoma sp.RFP). The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma.

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

scholarly journals Peptide-Based Formulation from Lactic Acid Bacteria Impairs the Pathogen Growth in Ananas Comosus (Pineapple)

Coatings ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 457 ◽  
Author(s):  
Gabriela N. Tenea ◽  
Daniela Olmedo ◽  
Clara Ortega
Keyword(s):  
Significant Proportion ◽  
High Capacity ◽  
Total Cell ◽  
Ananas Comosus ◽  
Street Vending ◽  
E Coli ◽  
Cell Counts ◽  
Total Cell Density

Worldwide, street vending commerce has grown exponentially, representing in some countries, including Ecuador, a significant proportion of food consumed by the urban population. Pineapple is one of the common fruits sold as ready-to-eat slices by ambulant vendors in the street or on public transport at risk of contamination by various microorganisms. Previously, we selected Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis Gt28 strains producing peptides with high capacity to inhibit pathogen growth in vitro. In this study, the effect of different edited formulations containing a mixture of Cys5-4/Gt28 peptides was evaluated in vitro and ex vitro against a pathogenic cocktail containing E. coli (2), Salmonella (2) and Shigella (1). The growth of bacterial cocktail co-inoculated with cell-free supernatant containing peptides (formulation T1) and precipitated peptides (formulation T6), in a ratio of Cys5-4/Gt28:1:1 (v/v), results in a decrease of total cell viability with 1.85 and 1.2 log CFU/mL orders of magnitude at 6 h of incubation. About the same decrease (1.9 log CFU/g) was observed when pineapple slices artificially inoculated with the pathogenic cocktail were coated with T1 formulation, indicating the capacity to diminish simultaneous pathogens in situ, thus demonstrating its great biological control and protection. However, the E. coli cell counts reduced by 2.08 log CFU/g while Salmonella and Shigella cell counts reduced by 1.43 and 1.91 log CFU/g, respectively, at 5 days of refrigeration. In the untreated pineapple slices, the total cell density was maintained during storage, suggesting the adaptation of the pathogens to the fruit matrix. The peptide-based formulation exerted a bacteriolytic mode of action inducing pathogenic cell death. The results indicate that coating pineapple slices with peptide-based formulation is a promising approach to protect them from further contamination by microbial spoilage as well as an alternative to increase the food safety.

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

scholarly journals Antimicrobial effects of fruit sauces on some pathogenic bacteria in vitro and on chicken breast meat

2021 ◽  
Vol 72 (1) ◽  
pp. 2703
Author(s):  
I VAR ◽  
S UZUNLU ◽  
I DEĞIRMENCI
Keyword(s):  
Antimicrobial Activity ◽  
Pathogenic Bacteria ◽  
Diffusion Method ◽  
Chicken Breast ◽  
Type I ◽  
Antimicrobial Effects ◽  
E Coli ◽  
Agar Diffusion

The use of natural food additives is currently a rising trend. In the present study, the aim was to determine the antimicrobial effects of plum, pomegranate, Seville orange and sumac sauces on E. coli O157:H7,E. coli type I,Listeriamonocytogenes, Listeria ivanovii, Salmonella Typhimurium and Staphylococcus aureus. Different concentrations (1%, 10%, 100%, v/v) of the sauces were tested on the studied bacteria in vitro using the agar diffusion and minimal inhibition concentration (MIC) methods. The results showed that the sumac sauce had the highest antimicrobial activity. The Seville orange, plum and pomegranate sauces also exerted antimicrobial activity in descending order. The antimicrobial activity of the fruit sauces was more effective at a concentration of 100% than at 10% and 1%, v/v. The most inhibitory effect was recorded for sumac sauce at a concentration of 100% (v/v) on L.monocytogenesand E. coli O157:H7. The findings of the MIC method aligned with the agar diffusion method. In addition, the in situ(food method) antimicrobial effect of the sauces on the indigenous microflora of chicken breast samples sold in stores was determined. Chicken samples hosting aerobic mesophilic bacteria, coliforms and E. coli were treated for two hours at 4 °C with plum, pomegranate, Seville orange and sumac sauces and were then monitored. The findings revealed that the Seville orange and sumac sauces were the most effective in reducing the indigenous microbial growth on the chicken samples. The plum sauce showed higher antimicrobial activity than pomegranate sauce. The phenolic content and acidity of the samples significantly (P< 0.05) affected the antimicrobial activity both in vitro (agar diffusion and MIC) and in situ (chilled chicken breast). In conclusion, the sumac and Seville orange sauces were found to be the most promising natural antibacterial agents, and their use could be recommended, for example, in catering services to reduce the risk of foodborne illness.

326 GENERATION OF A DOUBLE-TRANSGENIC PIG WITH PANCREAS-SPECIFIC GREEN AND LIVER-SPECIFIC RED FLUORESCENCE

2013 ◽  
Vol 25 (1) ◽  
pp. 311 ◽  
Author(s):  
H. Matsunari ◽  
K. Nakano ◽  
T. Kanai ◽  
R. Sakai ◽  
M. Watanabe ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Proteins ◽  
Green Fluorescence ◽  
Tissue Specific ◽  
Specific Fluorescence ◽  
Red Fluorescence ◽  
Feasible Option ◽  
Organ Tissue ◽  
Transfer Method

Transgenic (Tg) pigs with organ/tissue-specific fluorescence expression provide invaluable research tools for many types of studies, such as organogenesis analysis, in vitro tissue generation from pluripotent cells, and progenitor/stem cell transplantation therapy. We aimed to develop a Tg pig characterised by pancreas- and liver-specific fluorescence expression. A 8.4 kb transgene construct expressing Venus (green fluorescence) under the control of the mouse Pdx1 (pancreatic duodenal homeobox-1) promoter and a BAC-derived construct (170 kb) consisting of the whole-length porcine albumin (Alb) promoter and humanized Kusabira-Orange (huKO, red fluorescence) was introduced into porcine in vitro-matured oocytes using the intracytoplasmic sperm injection (ICSI)-mediated gene transfer method. Injected embryos were transferred to the oviducts of oestrus-synchronized recipients after culture for 1 to 3 days. The transfer of 370 Pdx1-Venus embryos into 4 recipients produce 22 (5.9%) fetuses/piglets, and 9 (40.9%) Tg pigs exhibited pancreas-specific Venus expression. Two (1 male and 1 female) founder Pdx1-Venus-Tg pigs were mated with wild-type (WT) pigs and produced 32 offspring in 3 litters, of which 16 (50.0%) were transgenic. Pancreas-specific Venus expression was inherited in these Tg offspring. The transfer of 523 Alb-huKO embryos into 4 recipients resulted in 19 (3.6%) piglets including a Tg female, which showed liver-specific huKO fluorescent expression. Expression of huKO was detected by RT-PCR exclusively in liver, but not in 7 other organs/tissues examined, including heart, lung, stomach, small intestine, spleen, kidney and skin. This founder Tg female produced a total of 12 non-Tg and 5 Tg offspring (in 2 litters) after mating with a WT boar. Liver-specific huKO expression was inherited in these Tg offspring. Furthermore, the mating of a female Pdx1-Venus pig with an Alb-huKO boar yielded 7 non-Tg and 10 Tg pigs. Four of these Tg pigs carrying both of the transgenes exhibited both pancreas-specific Venus and liver-specific huKO expression in single individuals. Double-Tg pigs with pancreas-specific green fluorescence and liver-specific red fluorescence grew normally, and tests of their reproduction ability are currently underway. These data demonstrate that transgene introduction by ICSI-mediated gene transfer into in vitro-matured oocytes is a feasible option for generating pigs expressing fluorescent proteins in a tissue-specific manner.

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