Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.     

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund–Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K_i ≤1 µM). The distinct inhibition of WRN and BLM helicases by the minor groove binders suggest that these helicases unwind double-stranded DNA by a related mechanism.     

Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein.     

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.     

Water use and water-use efficiency of coppice and seedling Eucalyptus globulus Labill.: a comparison of stand-scale water balance components

Aims

Growers of Eucalyptus globulus Labill. plantations can establish second and later rotations from coppice or by replanting with seedlings. At most locations where E. globulus is grown commercially, water availability is a major driver for productivity. Thus growers must consider which reestablishment technique will maximize productivity whilst sustaining site resources for subsequent rotations. In this study we aimed to compare the stand-scale water balance components of young coppice and seedling E. globulus.

Methods

A second rotation E. globulus coppice and seedling trial was monitored for two successive seasonal cycles. Coppice and seedling plots were instrumented with sap flow- and meteorological-sensors so that stand-scale water balance components could be estimated on a daily time step.

Results

Stand-scale transpiration rate (E) and rate of interception (E _I) were larger in coppice compared to seedlings, but the rate of soil evaporation (E _S) was lower. At approximately 2?years of age each coppice stump was reduced to a single dominant stem, a standard management practice for E. globulus growers, which reduced stem biomass by approximately 70% and caused E to fall to a value approximating that in seedlings. The cumulative transpiration of coppice was 425?mm greater than seedlings up to 34?months of age. Without the coppice reduction (down to one stem/stump), we estimate that the difference would have been much greater. The water-use efficiency of stem production (WUE_stem) was greater in young coppice compared to seedlings but this benefit is likely to be offset by the loss of biomass (and thus transpired water) during coppice stem reduction.

Conclusion

Under the conditions of this study, which included reducing coppice to a single stem, reestablishment with seedling E. globulus resulted in a higher water-use efficiency of stem biomass production compared to coppice of a similar age.     

Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and trzN Genes in Viral-Community DNA

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.