On the streets of San Francisco: highlights from the ISSCR Annual Meeting 2010

The 2010 Annual Meeting of the International Society for Stem Cell Research (ISSCR) was held in San Francisco in June with an exciting program covering a wealth of stem cell research from basic science to clinical research.     

Silvopastoralism in the Alps: Native plant species selection under different grazing pressure

To evaluate the suitability of wood pastures as a managing tool in subalpine regions it is essential to know more about the influence of grazing on the ground vegetation. This study assessed native plant species selection by cattle at different stocking rates, feeding habits and site preferences of cattle. Based on the results, conclusions concerning the value of silvopastoral systems in the Alps were drawn. A field study on six different wood pasture areas, grazed by cattle at different stocking rates, was accompanied by an experiment on three adjoining areas of 0.51 ha each, stocked with either three, six, or nine heifers. Plant species were recorded in plots of 20 cm × 20 cm before and after grazing, and the intensity of grazing on each species was assessed. At low stocking rates, grasses and tall species were most intensely grazed, while at higher stocking rates the intake of forbs and small species increased. Since no relationship was found between nutritional value and species preference, other factors such as accessibility of a plant seem to be important for the feeding preferences of cattle. The preference for grasses at low and medium stocking rates suggests that an increased growth of forbs might lead to an increase in plant species diversity.     

Postthaw survival of rhesus macaque sperm: variation in the response of individual males to different freezing protocols

This study was designed to assess the effects of various cryopreservation protocols on the postthaw survival of rhesus macaque sperm. Multiple ejaculates of five mature males were collected and frozen by each of four different methods. Cryopreservation significantly reduced motility in all samples, regardless of the method, and the response of different ejaculates of the same male to each method proved to be consistent. The freezing method was a significant factor in determining the immediate postthaw motility, although this effect disappeared after 4 hr. Moreover, there was a significant interaction between individual males and freezing method suggesting that developing a single freezing protocol that is universally suitable may be difficult.     

Mercury and non-protein thiol compounds in the seagrass Posidonia oceanica

Mercury concentrations, non-protein thiol levels and the enzyme activities of glutathione-S-transferase (GST) were measured in the blades and sheaths of the marine phanerogam Posidonia oceanica. The seagrass was collected in January and June and at three sites: the Bay of Rosignano (Italy) known for its mercury contamination, the north of the Lérins islands (Bay of Cannes, France), the Bay of Tonnara (Corsica, France). The two latter sites are considered as free of any known industrial inputs. Mercury concentrations and GST activities in both tissues were always higher in samples from Rosignano, particularly in June. Non-protein thiol levels were significantly higher in the blades than in the sheaths of P. oceanica from Tonnara and Lérins. In contrast, at Rosignano, the sheaths presented a significantly higher non-protein thiol concentration than the blades, particularly in June. Levels in the sheaths appeared to increase with the degree of pollution. Western Blot performed on sheaths of P. oceanica collected in June at Rosignano and Lérins revealed a characteristic band of GSTs at 31 kDa, proving the presence of the GST enzyme in this tissue. Mercury seemed to exert an influence upon non-protein thiol metabolism, including GST induction, in P. oceanica collected from the NW Mediterranean.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate‐dependent thioredoxin reductase

The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent thioredoxin reductase (NTR) in a multistep transfer of reducing equivalents from NADPH to Trx via a tightly NTR‐bound flavin. Here, interactions between NTR and Trx are predicted by molecular modelling of the barley NTR:Trx complex (HvNTR2:HvTrxh2) and probed by site directed mutagenesis. Enzyme kinetics analysis reveals mutants in a loop of the flavin adenine dinucleotide (FAD)‐binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent on these two residues, suggesting a distinct mode for NTR:Trx recognition. Comparison between the HvNTR2:HvTrxh2 model and the crystal structure of the Escherichia coli NTR:Trx complex reveals major differences in interactions involving the FAD‐ and NADPH‐binding domains as supported by our experiments. Overall, the findings suggest that NTR:Trx interactions in different biological systems are fine‐tuned by multiple intermolecular contacts. Proteins 2014; 82:607–619. © 2013 Wiley Periodicals, Inc.     

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.     

Requirement for the second coding exon of Tat in the optimal replication of macrophage-tropic HIV-1

HIV-1 Tat is essential for virus replication and is a potent transactivator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Here, we find that the second coding exon of Tat contributes a novel function for the replication/infectivity of macrophage-tropic HIV-1. We show that macrophage-tropic HIV-1 which expresses the full-length two-exon form of Tat replicates better in monocyte-derived macrophages (MDM) than an otherwise isogenic virus which expresses only the one-exon form of Tat. Similarly, two-exon Tat expressing HIV-1 also replicates better than one-exon Tat expressing HIV-1 in two different models of human cells/tissue reconstituted SCID mice.     

A novel form of oxytocin in New World monkeys

Oxytocin is widely believed to be present and structurally identical in all placental mammals. Here, we report that multiple species of New World monkeys possess a novel form of oxytocin, [P8] oxytocin. This mutation arises from a substitution of a leucine to a proline in amino acid position 8. Further analysis of this mutation in Saimiri sciureus (squirrel monkey) indicates that [P8] oxytocin is transcribed and translated properly. This mutation is specific to oxytocin, as the peptide sequence for arginine vasopressin, a structurally related nonapeptide, is unaltered. These findings dispel the notion that all placental mammals possess a 'universal' oxytocin sequence, and highlight the need for research on the functional significance of this novel nonapeptide in New World monkeys.     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.