Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-beta) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2-3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34(+) progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFbeta-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFbeta3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair.     

Involvement of abscisic acid-dependent and -independent pathways in the upregulation of antioxidant enzyme activity during NaCl stress in cotton callus tissue

The role of abscisic acid (ABA) in the signal transduction pathway associated with NaCl-induced up-regulation of antioxidant enzyme activity was examined in a NaCl-tolerant cotton callus cell line treated with NaCl, ABA, paraquat, or H₂O₂ in the presence and absence or fluridone, an inhibitor of terpene, and therefore, ABA synthesis. Treatment with NaCl resulted in a rapid increase (within 30 minutes) in the ABA levels of the callus tissue, and the NaCl, ABA, and paraquat treatments induced rapid increases in the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase. Pre-treatment with fluridone significantly suppressed the NaCl-induced increases, but only slightly delayed the increases in tissue subjected to exogenous ABA treatment. This implies that ABA is involved in the signal transduction pathway associated with the NaCl-induced up-regulation of these antioxidant enzymes. Pre-treatment with fluridone had no effect on the paraquat-induced increases, suggesting that these enzymes can also be up-regulated by a pathway other than the one mediated by ABA. Both the NaCl and paraquat treatments produced significant increases in the superoxide levels within the callus, but the increase resulting from the paraquat treatment was significantly higher than the increase resulting from the NaCl treatment. These data suggest that NaCl stress results in the production of reactive oxygen intermediates (ROI) which signals the induction of an ABA-dependent signaling pathway. The production of very high levels of ROI, such as those that occur with paraquat treatment or perhaps during periods of prolonged or extreme stress, may induce an ABA-independent signaling pathway.     

Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.     

Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome

Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.Ubiquitination is a highly specific process that involves a group of proteins responsible for adding ubiquitin molecules to cellular substrates, thereby resulting in the modulation of numerous cellular pathways.¹ The deregulation of components of the ubiquitin conjugation system causes defects in many cellular functions and these have been associated with human pathogenesis.² Of the components involved in the ubiquitin cascade, the E3 ubiquitin ligases provide the substrate specificity. By attaching ubiquitin molecules to their substrates, E3 ligases have direct control over the functions and, in many cases, protein turnover of these substrates. In addition, loss of function in a number of E3 enzymes has been shown to have an important role in the development of severe physiological conditions such as certain cancers and neurological disorders.³ A representative instance of the latter is Angelman syndrome (AS), a severe neurodevelopmental disorder, with clinical features of mental retardation, developmental delay, ataxia and epilepsy.^{4, 5} The principal protein affected in AS is the E3 ubiquitin ligase E6-associated protein (E6AP/UBE3A), the gene being found on chromosome 15q11-13. UBE3A was initially identified as an interacting partner of high-risk HPV-16 and -18 E6 oncoproteins,^{6, 7} but was subsequently found to be linked to the development of AS. AS develops mainly due to genetic defects that lead to the loss of expression of the maternal allele of the UBE3A gene in the hypothalamus.^{8, 9} Between 65 and 75% of AS patients have been diagnosed with the deletions of 15q11-13, 3–7% of patients show uniparental disomy and ~3% of cases have been found with imprinting defects, such that the functionally defective maternal copy of the gene is expressed in the brain.⁵ In addition, there are also 5–11% of individuals with AS whose sequence analyses show UBE3A mutations. Most of these have in-frame deletions that would be predicted to result in protein truncations,^{10, 11} but a number of those patients have milder mutations, such as point mutations, that do not affect the expression of the full-length protein.^{12, 13} The majority of these mutations however are defective in ubiquitin ligase activity, indicating that the loss of enzymatic activity of UBE3A is important in promoting the development of AS.¹⁴Studies have demonstrated that ubiquitin ligase activity of UBE3A has a role in the proteasome-dependent degradation of several cellular substrates, and it can be reasoned that defects in the regulation of some of these substrates can contribute to AS development. However, although a number of UBE3A target proteins have been identified, including Sox9, C/EBPα, α-Synuclein, p27, promyelocytic leukemia (PML) tumor suppressor, annexin A1, amplified in breast cancer 1 (AIB1) and HHR23A,^{15, 16, 17, 18, 19, 20, 21, 22} characterization of their interactions with UBE3A have only partially contributed to an understanding of the molecular mechanisms behind the development of AS pathology. In addition, UBE3A has also been shown to interact with other components of the proteasome degradatory pathway, including the ubiquitin ligases HERC2, Ring1B and EDD,^{23, 24, 25} and recent studies demonstrated a direct interaction between UBE3A and the proteasome itself.^{26, 27} Whether any of these interactions might also be involved in AS development is an open question. Thus, although many proteins are known to be targeted by UBE3A for proteasomal degradation, much less is known about UBE3A interactions with the proteasome itself, or how these interactions might affect substrate turnover, or whether perturbations in this association can contribute to AS development.The 26S proteasome is a complex cellular machine that contains a 20S central core, a hollow tube composed of multiple proteasome subunits, which contain proteolytic sites. On each end of the 20S proteolytic core, there is an ATP-dependent 19S regulatory particle, which is involved in capturing the ubiquitinated proteins.²⁸ Among several subunits that are part of the 19S regulatory particle complex, there are two major ubiquitin receptors, Rpn10/S5a and Rpn13.^{29, 30, 31} The S5a subunit mediates the targeting of ubiquitin substrates to the proteasome by binding ubiquitin conjugates through a ubiquitin-interacting motif (UIM)³² and loss of this activity of S5a results in decreased proteolytic activity of the proteasome.^{33, 34, 35} It has also been shown that S5a is regulated by mono-ubiquitination, which inhibits its ability to interact with ubiquitin-conjugated substrates, and also leads to decreased proteasome activity.³¹ Recent studies have shown that UBE3A can directly ubiquitinate the S5a subunit, and that its Drosophila ortholog, Ube3a, mediates ubiquitination of the Drosophila S5a homolog, resulting in its subsequent degradation.^{26, 27} Structural studies have indicated that a number of AS-associated UBE3A point mutations occur in the HECT domain, which most likely lead to the expression of catalytically defective proteins.^{13, 14} We were therefore interested in investigating whether catalytically defective AS-associated point mutants can still interact with the S5a subunit and, furthermore, in determining whether they can exert any inhibitory effects on the proteasomal turnover of ubiquitinated substrates. We show here that AS-associated UBE3A mutants interact more strongly with S5a, with one of the consequences being a general inhibitory effect on the overall proteolytic activity of the proteasome. These results suggest that perturbation of overall proteasome function may be an important element in the development of AS, which thus shows many similarities with other proteasomal neurogical defects.     

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions.     

Adult survival and microsatellite diversity in possums: effects of major histocompatibility complex-linked microsatellite diversity but not multilocus inbreeding estimators

Adult survival is perhaps the fitness parameter most important to population growth in long-lived species. Intrinsic and extrinsic covariates of survival are therefore likely to be important drivers of population dynamics. We used long-term mark-recapture data to identify genetic, individual and environmental covariates of local survival in a natural population of mountain brushtail possums (Trichosurus cunninghami). Rainfall and intra-individual diversity at microsatellite DNA markers were associated with increased local survival of adults and juveniles. We contrasted the performance of several microsatellite heterozygosity measures, including internal relatedness (IR), homozygosity by loci (HL) and the mean multilocus estimate of the squared difference in microsatellite allele sizes within an individual (mean d ²). However, the strongest effect on survival was not associated with multilocus microsatellite diversity (which would indicate a genome-wide inbreeding effect), but a subset of two loci. This included a major histocompatibility complex (MHC)-linked marker and a putatively neutral microsatellite locus. For both loci, diversity measures incorporating allele size information had stronger associations with survival than measures based on heterozygosity, whether or not allele frequency information was included (such as IR). Increased survival was apparent among heterozygotes at the MHC-linked locus, but the benefits of heterozygosity to survival were reduced in heterozygotes with larger differences in allele size. The effect of heterozygosity on fitness-related traits was supported by data on endoparasites in a subset of the individuals studied in this population. There was no apparent density dependence in survival, nor an effect of sex, age or immigrant status. Our findings suggest that in the apparent absence of inbreeding, variation at specific loci can generate strong associations between fitness and diversity at linked markers.     

EVIDENCE FOR XYLEM CONSTRICTIONS IN THE PRIMARY VASCULATURE OF PSILOPHYTON DAWSONII,AN EMSIAN TRIMEROPHYTE

Serial peels through the branch junctions of Psilophyton dawsonii were examined in an attempt to determine if “hydraulic constrictions” (i.e., a localized decrease in mean diameter of xylem elements) sensu Zimmermann (1983) had occurred during bifurcation. Based on the mean diameters of primary xylem tracheids measured acropetally and basipetally to branch junctions, evidence for xylem constrictions (= localized decrease in mean tracheid diameter) was found within the basalmost portions of four out of six minor axes examined near their attachment to major axes (anisotomous junctions). Xylem constrictions were not detected in branch junctions between axes of equal girth (isotomous junctions). Xylem constrictions were detected within the base of five out of seven junctions between fertile and main axes. The mean diameters of tracheids of the branch trace near its origin are larger both basipetally and acropetally from the constriction. There is no evidence for a localized decrease in the mean diameter of the xylem strand in the region of a constriction. Therefore, xylem constrictions are not the result of epidogenesis. Based on the production of “hydraulic constrictions” in extant plants, which serve to localize the formation of embolisms in lateral branches during water stress, it is speculated that P. dawsonii could protect the vasculature of major axes by a similar anatomical feature.     

Effects of plot vegetation diversity and spatial scale on Coccinella septempunctata movement in the absence of prey

The influence that vegetation diversity and the spatial scale of that diversity exert on insect behavior has increasingly been explored in the ecological literature, but relatively few experiments have explicitly incorporated both factors in experimental treatments. We conducted a field study designed to explore the effect of both of these factors on insect movement behavior in a broccoli agroecosystem. We caught and released seven‐spotted ladybird beetles (Coccinella septempunctata L.) in plots containing different degrees of vegetation diversity at two different spatial scales in which prey had been removed. Beetle movement was recorded at timed intervals, and move lengths and turning angles were used to generate discrete path maps for each beetle. Observed mean beetle net squared displacements were compared with predicted net squared displacements, and 95% confidence intervals were generated using a bootstrap method described by Turchin (1998 ) [Quantitative Analysis of Movement: Measuring and Modeling Population Redistribution in Animals and Plants. Sinauer Associates Inc., Sunderland, MA.]. Predicted net squared displacements underestimated beetle movement in smaller plots with both low and higher vegetation diversity for the first five move lengths, whereas no significant difference between observed and predicted net squared displacement for beetles in larger plots of either level of vegetation diversity were detected. These findings highlight the need for a better understanding of how natural enemies are influenced by vegetation diversity and the spatial scale of that vegetation in agroecosystems. The implications of these results for biological control are discussed.     

Effects of radiograph projection parameter uncertainty on TKA kinematics from model-image registration

Model-image registration techniques have been used extensively for the measurement of joint kinematics in vivo. These techniques typically utilize an explicit measurement of X-ray projection parameters (principal distance, principal point), which is easily done for prospective studies. However, there is vast opportunity to derive useful information from previously collected clinical radiographic films where the projection parameters are unknown. The purpose of this study was to determine variation in measured knee arthroplasty kinematics when the X-ray projection parameters were unknown, but bounded. Based on the clinical radiographic protocol, a nominal principal point was chosen and eight additional points ±2 and ±5 cm in the horizontal and vertical directions were defined. Tibiofemoral kinematics were determined for all nine projection parameter sets for a series of 10 lateral radiographs. In addition, the principal distance was varied ±15 cm and tibiofemoral kinematics were determined for these two projection sets. Measured joint kinematics varied less than 0.6° and 0.4 mm for ±2 cm variations in principal point location, and 0.7° and 0.6 mm for ±5 cm variations in principal point location. Measured joint kinematics varied less than 0.6° and 0.7 mm for ±15 cm variations in principal distance. Variation in X-ray principal point and principal distance over clinically bounded ranges has a small effect on knee arthroplasty kinematics computed from model-image registration with high-quality clinical radiographs.     

Temporal and spatial responses of Chironomidae (Diptera) and other benthic invertebrates to urban stormwater runoff

In a longitudinal study of two streams whose lower reaches received unattenuated urban stormwater runoff, physical disturbance by stormflow was less important than the persistant unidentified chemical impacts of urban stormwater in limiting the distribution of Chironomidae, and Ephemeroptera, Trichoptera and Plecoptera (EPT). A hierarchical spatial analysis showed that chironomid density did not decrease from rural to urban stream reaches. Instead, the taxonomic composition of chironomid assemblages was significantly altered in urban versus rural reaches; chironomid assemblages in urban reaches exhibited higher average pollution tolerance scores. In contrast, the density of EPT was significantly lower in urban reaches. Despite higher values of stormflow tractive force in urban reaches, streambed stability tended to be greater in urban reaches. Modeling of temporal variation in chironomid density showed similar patterns in both rural and urban reaches: chironomid density had a unimodal relationship to rainfall index (RI), with highest densities at intermediate values of RI. Models of EPT density over time in rural reaches showed no significant relation to RI, and temporal variation in EPT density in urban reaches was not predictable. The abundance of fine particulate organic matter, including periphyton (FPOM), on cobbles was greater in urban reaches and showed a much greater degree of temporal variation than in rural reaches. In urban reaches, a negative relation between FPOM and RI indicated the importance of stormflow abrasion. Handling editor: K. Martens