The zinc finger protein Ynr046w is plurifunctional and a component of the eRF1 methyltransferase in yeast

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 A resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three alpha-helices. The active methyltransferase is the heterodimer Ydr140w.Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.     

Global health policies that support the use of banked donor human milk: a human rights issue

Background

The past ten years have witnessed a rising trend in the prevalence and duration of breastfeeding in Italy, but breastfeeding rates increase in an unequal way; they are higher in the North of Italy than in the South. The purpose of this study was to describe the experiences, expectations and beliefs of a sample of mothers, and to identify differences, if any, between the North and the South of Italy.

Methods

The study was conducted in two regions of Italy, Friuli Venezia Giulia in the Northeast and Basilicata in the South. Two hundred and seventy-nine mothers of infants and children 6 to 23 months of age were interviewed using an 85-item questionnaire including closed and open questions on infant feeding experiences and beliefs, sources of information and support, reasons for intended and actual choices and practices, and some demographic and social variables. Face-to-face interviews were conducted between May 2001 and September 2002. Quantitative and qualitative methods were used for data analysis.

Results

The distribution of the mothers by age, education, employment and parity did not differ from that of the general population of the two regions. The reported rates of initiation and duration of breastfeeding were also similar: 95% started breastfeeding, exclusive breastfeeding was 32% at three and 9% at six months, with 64% and 35% of any breastfeeding, respectively. Some differences were reported in the rates of full breastfeeding, reflecting different ages of introduction of non-nutritive fluids. These, as well as nutritive fluids – including infant formula – and complementary foods, were introduced far too early. Advice on infant feeding was generally provided by health professionals and often was not based on up-to-date recommendations. Mothers were generally aware of the advantages of breastfeeding, but at the same time reported problems that they were not able to solve alone or through social and health system support. Most mothers would welcome the support of a peer counsellor. More mothers in Basilicata than in Friuli Venezia Giulia reported difficulties with breastfeeding related to returning to work and were not familiar with their rights on breastfeeding and maternity leave.

Conclusion

Programmes for the protection, promotion and support of breastfeeding in these and similar regions of Italy should concentrate on better training of health professionals with regards to lactation management, communication, and counselling skills. The addition of trained peer counsellors could reinforce the work done by the health system and, through community involvement, could help change social prejudice in the mid- and long-term. The differences between regions should be taken into account in formulating these programmes to avoid increasing, and possibly to decrease, the current gaps.     

Functional genomics: applying calcium imaging and RNA interference to Drosophila cell lines to identify new roles for gene products

The selective knockdown of genes using RNA interference, and the analysis of the resultant phenotype using calcium imaging, is proving to be a powerful combination for discovering new genes involved in calcium signalling. This technical note describes the application of this approach to the Drosophila S2 cell line and its derivatives.     

A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart

Key molecules which regulate the formation of the heart have been identified; however, the mechanism of cardiac morphogenesis remains poorly understood at the cellular level. We have adopted a genetic approach, which permits retrospective clonal analysis of myocardial cells in the mouse embryo, based on the targeting of an nlaacZ reporter to the alpha-cardiac actin gene. A rare intragenic recombination event leads to a clone of beta-galactosidase-positive myocardial cells. Analysis of clones at different developmental stages demonstrates that myocardial cells and their precursors follow a proliferative mode of growth, rather than a stem cell mode, with an initial dispersive phase, followed by coherent cell growth. Clusters of cells are dispersed along the venous-arterial axis of the heart tube. Coherent growth is oriented locally, with a main axis, which corresponds to the elongation of the cluster, and rows of cells, which form secondary axes. The angle between the primary and secondary axes varies, indicating independent events of growth orientation. At later stages, as the ventricular wall thickens, wedge shaped clusters traverse the wall and contain rows of cells at a progressive angle to each other. The cellular organisation of the myocardium appears to prefigure myofibre architecture. We discuss how the characteristics of myocardial cell growth, which we describe, underlie the formation of the heart tube and its subsequent regionalised expansion.     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.     

The mouse as a model of heart morphogenesis in mammals: origin and lineage of myocytes

In order to follow cardiac precursor cells, we have adopted a retrospective clonal approach, based on the nlaacZ genetic label. Random clones were generated and observed at different developmental stages in murine myocardium. The distribution of these clones in clusters suggest for the first time that cells fated to form myocardium proliferate in two steps. The first growth phase, before E8.5, is dispersive and polarised along the axis of the primitive cardiac tube, contributing to its elongation. The second growth phase is coherent and polarised differentially in different cardiac subregions. Interestingly, this can be correlated with production of geometrical forms (dilatation of a sphere, enlargement of a tube), showing the relation between heart morphogenesis and the controlled proliferation of myocardial cells. The restricted distribution of clones to the right or left ventricule was also investigated with the goal of establishing the time at which cardiac chamber identity emerges. Right and left ventricular lineages appear to segregate early, in agreement with the existence of two populations of cardiac precursors, the so-called primary (or posterior) and secondary (or anterior) heart fields.     

Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein

A major group of colicins comprises molecules that possess nuclease activity and kill sensitive cells by cleaving RNA or DNA. Recent data open the possibility that the tRNase colicin D, the rRNase colicin E3 and the DNase colicin E7 undergo proteolytic processing, such that only the C-terminal domain of the molecule, carrying the nuclease activity, enters the cytoplasm. The proteases responsible for the proteolytic processing remain unidentified. In the case of colicin D, the characterization of a colicin D-resistant mutant shows that the inner membrane protease LepB is involved in colicin D toxicity, but is not solely responsible for the cleavage of colicin D. The lepB mutant resistant to colicin D remains sensitive to other colicins tested (B, E1, E3 and E2), and the mutant protease retains activity towards its normal substrates. The cleavage of colicin D observed in vitro releases a C-terminal fragment retaining tRNase activity, and occurs in a region of the amino acid sequence that is conserved in other nuclease colicins, suggesting that they may also require a processing step for their cytotoxicity. The immunity proteins of both colicins D and E3 appear to have a dual role, protecting the colicin molecule against proteolytic cleavage and inhibiting the nuclease activity of the colicin. The possibility that processing is an essential step common to cell killing by all nuclease colicins, and that the immunity protein must be removed from the colicin prior to processing, is discussed.     

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.