Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Additional analysis of dendrochemical data of Fallon, Nevada

Previously reported dendrochemical data showed temporal variability in concentration of tungsten (W) and cobalt (Co) in tree rings of Fallon, Nevada, US. Criticism of this work questioned the use of the Mann-Whitney test for determining change in element concentrations. Here, we demonstrate that Mann-Whitney is appropriate for comparing background element concentrations to possibly elevated concentrations in environmental media. Given that Mann-Whitney tests for differences in shapes of distributions, inter-tree variability (e.g., "coefficient of median variation") was calculated for each measured element across trees within subsites and time periods. For W and Co, the metals of highest interest in Fallon, inter-tree variability was always higher within versus outside of Fallon. For calibration purposes, this entire analysis was repeated at a different town, Sweet Home, Oregon, which has a known tungsten-powder facility, and inter-tree variability of W in tree rings confirmed the establishment date of that facility. Mann-Whitney testing of simulated data also confirmed its appropriateness for analysis of data affected by point-source contamination. This research adds important new dimensions to dendrochemistry of point-source contamination by adding analysis of inter-tree variability to analysis of central tendency. Fallon remains distinctive by a temporal increase in W beginning by the mid 1990s and by elevated Co since at least the early 1990s, as well as by high inter-tree variability for W and Co relative to comparison towns.     

Investigation of germination and aging in Moravian III barley grain by nuclear magnetic resonance.

High-resolution, solid-state 1H nuclear magnetic resonance (NMR) techniques are used for the first time to study germination in imbibed Moravian III barley grains. Whereas magic-angle spinning 1H NMR spectra reveal the water and lipid components in barley grains, combined rotation and multiple-pulse spectroscopy techniques provide 1H NMR spectra of grains that reveal the protein and carbohydrate as well as the water and lipid components. Spectra of grains are compared with spectra of model compounds to verify assignments. 1H T1 and T2 measurements using magic-angle spinning only and combined rotation and multiple-pulse spectroscopy techniques provide information about molecular mobility within the grains during inhibition. Some grains were subjected to artificial aging conditions. 1H NMR spectral comparisons are made between normal, viable grains and artificially aged grains.     

Is there a risk to living large? Large size correlates with reduced growth when stressed for knapweed populations

A central hypothesis in ecology is that plant life history evolution is constrained by fundamental “compromises between the conflicting selection pressures resulting from particular combinations of competition, stress, and disturbance”, with stress being defined as abiotic conditions that restrict production. Biogeographic differences among native and non-native ranges of invasive plants may provide unique opportunities for tests of this theory. We conducted a greenhouse experiment with Centaurea stoebe plants from North American and European populations. We compared the total biomass and phenotypic plasticity indices for plants from the native and non-native ranges under stressed and non-stressed conditions. The average size of Centaurea stoebe plants from 13 North American populations was greater than that of plants from 18 European populations regardless of stress treatment. However, when plants from the same populations were exposed to lower resources the differences in biomass between plants from North American and European populations were significantly less, suggesting that large plants were poorer stress tolerators. For all 31 populations the regression slope for the relationship between mean mass for populations in non-stressful conditions and mean mass in stressful conditions was less than 1.0, indicating that populations that produced large plants in good conditions also produced plants that grew disproportionately less in stressful conditions. These findings suggest that Centaurea stoebe may be evolving towards being a good “competitor” (sensu Grime, 1977) in its invasive range, but at the cost of being a good “stress tolerator”.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Regulation of Stomatal Tropism and Infection by Light in Cercospora zeae-maydis: Evidence for Coordinated Host/Pathogen Responses to Photoperiod?

Cercospora zeae-maydis causes gray leaf spot of maize, which has become one of the most widespread and destructive diseases of maize in the world. C. zeae-maydis infects leaves through stomata, which is predicated on the ability of the pathogen to perceive stomata and reorient growth accordingly. In this study, the discovery that light was required for C. zeae-maydis to perceive stomata and infect leaves led to the identification of CRP1, a gene encoding a putative blue-light photoreceptor homologous to White Collar-1 (WC-1) of Neurospora crassa. Disrupting CRP1 via homologous recombination revealed roles in multiple aspects of pathogenesis, including tropism of hyphae to stomata, the formation of appressoria, conidiation, and the biosynthesis of cercosporin. CRP1 was also required for photoreactivation after lethal doses of UV exposure. Intriguingly, putative orthologs of CRP1 are central regulators of circadian clocks in other filamentous fungi, raising the possibility that C. zeae-maydis uses light as a key environmental input to coordinate pathogenesis with maize photoperiodic responses. This study identified a novel molecular mechanism underlying stomatal tropism in a foliar fungal pathogen, provides specific insight into how light regulates pathogenesis in C. zeae-maydis, and establishes a genetic framework for the molecular dissection of infection via stomata and the integration of host and pathogen responses to photoperiod.     

Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition

S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.     

Oxalate contributes to the resistance of Gaillardia grandiflora and Lupinus sericeus to a phytotoxin produced by Centaurea maculosa

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (±)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardia grandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.