Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.     

The nitrogen handling characteristics of white clover (Trifolium repens L.) cultivars and a perennial ryegrass (Lolium perenne L.) cultivar

Ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) have contrasting responses to soil mineral N availability and clover has the ability to fix atmospheric N(2) symbiotically. It has been hypothesized that these differences are the key to understanding grass-clover coexistence and vegetative dynamics in pastures. However, the whole plant response of clover and ryegrass to mineral N availability has not been fully characterized and inter-cultivar variability in the N-handling dynamics of clover has not been assessed. A detailed experimental study to address these issues was undertaken. For all clover cultivars and ryegrass, mass specific mineral N uptake rates (of whole plants) were similar saturating functions of mineral N availability. For all clover cultivars total N assimilation rates, whole plant C : N ratios and root : shoot ratios were independent of mineral N availability. Clover growth rates were also independent of mineral N availability except for a slight (<10%) reduction at very low N availability levels. Specific N(2) fixation rate (whole plant) was precisely controlled to ensure fixation balanced the deficit between mineral N uptake and the total N assimilation required to maintain constant whole plant C : N ratio. There was always a deficit between N uptake and the total N assimilation required to maintain C : N ratio. Consequently, some N(2) fixation remained engaged even at high mineral N availability levels. All inter-cultivar variation in N(2) fixation dynamics could be attributed to variations in growth rate. Clover mass specific growth rate declined as plant size increased. Ryegrass specific growth rate, whole plant C : N ratio and root : shoot ratio were dependent on N availability. Increased N availability led to increased growth rate and decreased C : N and root : shoot ratios. Specific growth rate was also dependent on plant size, growth rate declining as plant size increased. It is concluded that clover inter-cultivar variation in field performance is unlikely to be a consequence of variation in N-handling characteristics. Inter-cultivar differences in growth rate are likely to be a much more important source of variation.     

Ligand design for alpha1 adrenoceptor subtype selective antagonists

Alpha1 adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring D analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human alpha1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (alpha1A, alpha1B, alpha1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the alpha1D adrenoceptor subtype is also reported.     

Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.     

High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the urokinase receptor in human glioma cells

The distribution of the urokinase-type plasminogen activator receptor (uPAR) on human glioma cells was examined as a function of culture conditions, using immunofluorescence and immunophotoelectron microscopy. Both uPAR colocalization with focal adhesion proteins and glioma cell motility were maximal in medium containing whole serum or a serum fraction retained by a 500,000 mol wt cutoff centrifugal concentration filter. High motility also took place in medium containing a serum fraction passed by the 500,000 cutoff filter but retained by a 100,000 cutoff filter and in minimal medium containing added vitronectin; however, under these conditions only a small percentage of the otherwise abundant focal adhesions contained colocalized uPAR. Glioma cells in minimal medium with added laminin migrated with a highly elongated morphology but without either classical focal adhesions or well-defined uPAR labeling. In contrast, glioma cells in minimal medium with no additions did not migrate, nor did they adhere well or display defined labeling patterns for focal adhesion proteins or uPAR. The results indicate that high-molecular-weight serum protein complexes promote both uPAR-focal adhesion colocalization and cell migration in glioma cells. However, conditions can be selected in which migration takes place with minimal uPAR-focal adhesion localization, as well as in the absence of apparent focal adhesions.     

Mineral concentration in selected native temperate grasses with potential use as biofuel feedstock

Stands of native grasses along roadways, in buffer strips, riparian zones and grass prairies have potential utility as feedstock for bioenergy production. The sustainability of harvesting these stands is reliant, in part, on knowledge of the mineral concentration of the harvested grasses because removal of mineral nutrients such as phosphorus (P) and potassium (K) can impact subsequent biomass production and ecosystem services associated with these stands. Mineral content of biomass, particularly that of silicon (Si), chlorine (Cl), and sulfur (S) also impacts thermochemical conversion approaches that convert grasses into bioproducts. This study quantified Cl, S, Si, P and K in Bromus marginatus, Elymus glaucus, Poa secunda, Pseudoroegneria, Elymus lanceolatus, Elymus trachycaulus, Leymus cinereus, Leymus triticoides, and Pseudoroegneria spicata collected at three growth developmental stages from four plant introduction stations located in the western US. Differences (P ? 0.05) in mineral concentrations were associated with developmental stage, species, and location. Variability was greatest in Si concentrations which ranged from 1847 to 28620 mg kg⁻¹, similar to those recorded in other grasses. Variability in Cl and S concentrations also occurred, but at less magnitude than that of Si. Concentrations of P and K, two mineral fertilizer components, varied approximately threefold among these grasses. Differences in mineral concentrations among these grasses were not completely dependent upon soil mineral content. Long-term evaluations of available soil mineral concentrations under contrasting management practices are needed to quantify how local conditions impact mineral cycling, and in turn, the sustainability of harvesting these stands. The data presented here establish baselines for these species in locations subject to contrasting environmental and microbiological conditions that affect mineral cycling and availability.     

Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (βTCP) via Fusion to a High Affinity,Multimeric βTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors

Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant βTCP scaffolds and to demonstrate the bioactivity of EGF tethered to βTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against βTCP and composites of βTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for βTCP. When a single copy of this βTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for βTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to βTCP, with only 25% of the protein released after 7 days at 37^oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance the proliferation of hBMSC when presented in tethered form on commercial βTCP bone regeneration scaffolds.