Non-integrative lentivirus drives high-frequency cre-mediated cassette exchange in human cells

Recombinase mediated cassette exchange (RMCE) is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening.     

Activation of wild type p53 function by its mortalin-binding, cytoplasmically localizing carboxyl terminus peptides

The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells.     

Apolipoprotein E polymorphism is related to plasma lipids and apolipoproteins in Mexican adolescents

Previous studies in the Mexican population have failed to show an effect of apolipoprotein E (APOE) polymorphism on the lipid profile. The purpose of the present study was to determine the frequencies of APOE phenotypes, and their influence on lipid and apolipoprotein levels in a random sample of Mexican adolescents living in Mexico City. APOE polymorphism, fasting insulin levels, lipid levels, and apolipoprotein levels were determined in 420 adolescents. We found a high frequency of APOE*3 subjects (89.5%) and a low frequency of APOE*2 (3.0%) and APOE*4 (7.5%) subjects. The APOE*4 subjects (including APOE 4,3 and APOE 4,4) showed the highest concentrations of total cholesterol, low-density lipoprotein cholesterol, and apoB and the lowest high-density lipoprotein cholesterol levels, whereas carriers of the APOE*2 allele (APOE 3,2 and APOE 2,2) had the lowest values for total and low-density lipoprotein cholesterol and the highest concentrations of high-density lipoprotein cholesterol. No significant differences in triglyceride and insulin levels among subjects with different APOE polymorphisms were observed. Unlike previous studies in the Mexican population, our results show that lipid and lipoprotein levels are under the influence of APOE polymorphism. As in whites, APOE*4 may be a cardiovascular risk factor in the Mexican population.     

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.     

Overexpression of NRPS4 leads to increased surface hydrophobicity in fusarium graminearum

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.