Lack of RNA-DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryos

There are numerous reports of the use of RNA-DNA oligonucleotides (chimeraplasts) to correct point mutations in vitro and in vivo, including the human apolipoprotein E gene (ApoE). Despite the absence of selection for targeting, high efficiency conversion has been reported. Although mainly used to revert deleterious mutations for gene therapy applications, successful use of this approach would have the potential to greatly facilitate the production of defined mutations in mice and other species. We have attempted to create a point mutation in the mouse ApoE gene by microinjection of chimeraplast into the pronuclei of 1-cell mouse eggs. Following transfer of microinjected eggs we analysed 139 E12.5 embryos, but obtained no evidence for successful conversion.     

The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function

Despite the rising knowledge about ribosome function and structure and how ribosomal subunits assemble in vitro in bacteria, the in vivo role of many ribosomal proteins remains obscure both in pro- and eukaryotes. Our systematic analysis of yeast ribosomal proteins (r-proteins) of the small subunit revealed that most eukaryotic r-proteins fulfill different roles in ribosome biogenesis, making them indispensable for growth. Different r-proteins control distinct steps of nuclear and cytoplasmic pre-18S rRNA processing and, thus, ensure that only properly assembled ribosomes become engaged in translation. Comparative analysis of dynamic and steady-state maturation assays revealed that several r-proteins are required for efficient nuclear export of pre-18S rRNA, suggesting that they form an interaction platform with the export machinery. In contrast, the presence of other r-proteins is mainly required before nuclear export is initiated. Our studies draw a correlation between the in vitro assembly, structural localization, and in vivo function of r-proteins.     

Covalent protein immobilization on glass surfaces: application to alkaline phosphatase

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4+/-0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.     

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.     

Characterization of the C2 subdomain of yeast mitochondrial initiation factor 2

The COOH-terminal part of the yeast mitochondrial initiation factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl-Met-tRNA(f)(Met) and unformylated Met-tRNA(f)(Met) with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements. The binding of coumarin-Met-tRNA(f) to either protein caused a blue shift of the coumarin emission spectrum and an increase in anisotropy. Full-length ymIF2 is functionally competent in forming an initiation complex and supporting formation of the first peptide bond on Escherichia coli ribosomes. The results demonstrate that ymIF2 has the same domain structure and biochemical properties of a typical IF2 species as found in bacteria or mammalian mitochondria--but with enhanced ability to bind unformylated initiator Met-tRNA.     

Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.