Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines

A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/-) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human A gamma- and epsilon-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery.     

Is Visual Selective Attention in Deaf Individuals Enhanced or Deficient? The Case of the Useful Field of View

Background

Early deafness leads to enhanced attention in the visual periphery. Yet, whether this enhancement confers advantages in everyday life remains unknown, as deaf individuals have been shown to be more distracted by irrelevant information in the periphery than their hearing peers. Here, we show that, in a complex attentional task, a performance advantage results for deaf individuals.

Methodology/Principal Findings

We employed the Useful Field of View (UFOV) which requires central target identification concurrent with peripheral target localization in the presence of distractors – a divided, selective attention task. First, the comparison of deaf and hearing adults with or without sign language skills establishes that deafness and not sign language use drives UFOV enhancement. Second, UFOV performance was enhanced in deaf children, but only after 11 years of age.

Conclusions/Significance

This work demonstrates that, following early auditory deprivation, visual attention resources toward the periphery slowly get augmented to eventually result in a clear behavioral advantage by pre-adolescence on a selective visual attention task.     

Membrene turnover in imbibed and dormant embryos of the wild oat (Avena fatua L.)

A comparative study of protein synthesis has been carried out with embryos excised from dormant (D) and non-dormant (ND) caryopses of the wild oat. Although D embryos imbibed in water or ND embryos imbibed in abscisic acid do not germinate, they incorporate [¹⁴C]leucine into TCA-insoluble material for the first 48 h as readily as embryos that do germinate (ND embryos imbibed in water, or D embryos imbibed in gibberellic acid). Pulsechase experiments with [¹⁴]leucine show that in both D and ND embryos the proteins associated with the membranes undergo turnover. The rates of decay of incorporated radioactivity are similar in both dormant and germinating embryos up to 98 h following embryo excision. Fractionation of the membrane proteins in SDS-polyacrylamide gels indicates that the different polypeptides have different rates of turnover. It is concluded that membrane proteins in imbibed D embryos are in a state of constant turnover, and that this is a part of the replacement processes necessary to maintain the integrity of hydrated cells. The continuation of such synthetic events could account for long term survival of dormant Avena fatua in the imbibed state.Abbreviations CCRSE cytochrome relative stain equivalents - D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid GA₃     

The rapidly evolving field of plant centromeres

Meiotic and mitotic chromosome segregation are highly conserved in eukaryotic organisms, yet centromeres--the chromosomal sites that mediate segregation--evolve extremely rapidly. Plant centromeres have DNA elements that are shared across species, yet they diverge rapidly through large- and small-scale changes. Over evolutionary time-scales, centromeres migrate to non-centromeric regions and, in plants, heterochromatic knobs can acquire centromere activity. Discerning the functional significance of these changes will require comparative analyses of closely related species. Combined with functional assays, continued efforts in plant genomics will uncover key DNA elements that allow centromeres to retain their role in chromosome segregation while allowing rapid evolution.     

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.     

Endocrine correlates of sexual behavior in the Mohor gazelle (Gazella dama mhorr)

In this study, we quantitatively examined male sexual behavior in relation to fecal estrogen and progesterone concentrations in female Mohor gazelles. We investigated the hypothesis that, during natural mating, males detect cues relating to the potential for successful conception and pregnancy. Time series analysis revealed that males could detect the approach of estrus 2-3 days before female fecal estrogens and estrogen/progestagen (E/P) ratio reached their peak values. Males also paid closer attention to those females excreting higher fecal estrogen concentrations. Mounting and copulation frequencies were positively correlated with both peri-ovulatory fecal estrogen concentrations, and the frequency of pre-copulatory courtship behaviors. These data suggested that males invest their reproductive effort selectively by mating the most fertile females, assuming that estrogen is a valid index of fertility. This assumption was investigated by examining sequential phases of the reproductive cycle for evidence that oocytes and follicles produced in a more estrogenic environment would lead to the formation of the most competent corpora lutea, thereby maximizing the chance of sustaining pregnancy. Associations between sexual behavior and hormone excretion support the hypothesis that males may use this mechanism to assess female fertility.     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels

During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg; the signals that mediate this process are poorly understood. Here, we describe a role for gamma-amino butyric acid (GABA) in pollen tube growth and guidance. In vitro, GABA stimulates pollen tube growth, although vast excesses are inhibitory. The Arabidopsis POP2 gene encodes a transaminase that degrades GABA and contributes to the formation of a gradient leading up to the micropyle. pop2 flowers accumulate GABA, and the growth of many pop2 pollen tubes is arrested, consistent with their in vitro GABA hypersensitivity. Some pop2 tubes continue to grow toward ovules, yet they are misguided, presumably because they target ectopic GABA on the ovule surface. Interestingly, wild-type tubes exhibit normal growth and guidance in pop2 pistils, perhaps by degrading excess GABA and sharpening the gradient leading to the micropyle.