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.     

Whole-genome scan, in a complex disease, using 11,245 single-nucleotide polymorphisms: comparison with microsatellites

Despite the theoretical evidence of the utility of single-nucleotide polymorphisms (SNPs) for linkage analysis, no whole-genome scans of a complex disease have yet been published to directly compare SNPs with microsatellites. Here, we describe a whole-genome screen of 157 families with multiple cases of rheumatoid arthritis (RA), performed using 11,245 genomewide SNPs. The results were compared with those from a 10-cM microsatellite scan in the same cohort. The SNP analysis detected HLA*DRB1, the major RA susceptibility locus (P=.00004), with a linkage interval of 31 cM, compared with a 50-cM linkage interval detected by the microsatellite scan. In addition, four loci were detected at a nominal significance level (P<.05) in the SNP linkage analysis; these were not observed in the microsatellite scan. We demonstrate that variation in information content was the main factor contributing to observed differences in the two scans, with the SNPs providing significantly higher information content than the microsatellites. Reducing the number of SNPs in the marker set to 3,300 (1-cM spacing) caused several loci to drop below nominal significance levels, suggesting that decreases in information content can have significant effects on linkage results. In contrast, differences in maps employed in the analysis, the low detectable rate of genotyping error, and the presence of moderate linkage disequilibrium between markers did not significantly affect the results. We have demonstrated the utility of a dense SNP map for performing linkage analysis in a late-age-at-onset disease, where DNA from parents is not always available. The high SNP density allows loci to be defined more precisely and provides a partial scaffold for association studies, substantially reducing the resource requirement for gene-mapping studies.     

Landscape-scale variation in the seed banks of floodplain wetlands with contrasting hydrology in China

1. At a local scale, the species composition, diversity and spatial variation of wetland plant communities are determined primarily by spatial and temporal heterogeneity in their environments. Less is known about variation at a landscape‐level. The floodplain of the Changjiang (Yangtze) River in China includes hydrologically connected, subtropical wetlands with different hydrological characteristics. 2. We examined seed‐bank species composition and richness in marshes of two contrasting hydrological types: permanent marshes, fed by local runoff, and lakeshore marshes more closely connected to the regulated river. Lakeshore marshes are flooded annually to depth of approximately 1 m and during flooding they support an alternate, aquatic vegetation type. The soil seed bank in March was a comparative estimator of species diversity. At the beginning of the growing season it included seeds from both phases of alternating vegetation types associated with the annual hydrological cycle. 3. A regional pool of 101 species was detected in the seed banks of six wetlands associated with the river and its tributaries: 56 occurred in permanent marshes and 59 in lakeshore marshes, with only 15 common to both. Species rarefaction curves indicated that more species occurred in permanent than lakeshore marshes at equal numbers of individuals sampled. However, the more heterogeneous lakeshore seed banks were estimated (Chao 2) to have greater total species richness (81) than permanent marsh (60). 4. Analysis using Sørensen's coefficient of similarity and DCA ordination revealed complex variation, with much greater differences between hydrological types than within them, irrespective of geographical distance. The types also differed significantly in the composition of four functional groups of species. 5. Despite the potential for dispersal of propagules via the annually pulsing river system (hydrochory), at a regional and landscape scale, diversity is maintained largely by large‐scale temporal hydrological heterogeneity and smaller scale spatial and topographic heterogeneity.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.     

Association of the FCRL3 gene with rheumatoid arthritis: a further example of population specificity?

Association of a functional promoter polymorphism mapping to the Fc receptor-like 3 (FCRL3) gene has recently been reported and replicated with rheumatoid arthritis (RA) in Japanese populations. The aim of this study was to investigate association of the FCRL3 gene with RA in UK subjects. DNA was available from 1065 patients with RA and 2073 population controls from the UK. Four single nucleotide polymorphism (SNP) markers (FCRL3-169*C/T (fclr3_3, rs7528684), fclr3_4 (rs11264799), fclr3_5 (rs945635), fclr3_6 (rs3761959)) all previously associated with RA in a Japanese population were genotyped in 761 RA samples and 484 controls. In the remaining samples, only the putative disease causal polymorphism, FCRL3-169*C/T, was tested. Genotyping was performed using either the Sequenom MassArray iPlex platform or a 5' Allelic discrimination assay (Taqman, ABI). Extensive linkage disequilibrium was present across the promoter SNPs genotyped (r² values = 0.60-0.98). Allele frequencies did not differ between RA cases and controls either for the putative disease causal polymorphism (odds ratio FCRL3-169*C allele = 0.97 (0.87-1.07), p = 0.51) or for the other SNPs tested. Similarly, no association was detected with RA using haplotype analysis or when stratification by shared epitope carriage or by presence of rheumatoid factor was undertaken. This study was powered to detect an effect size of 1.24 or greater for the FCRL3-169*C/T functional promoter polymorphism but no evidence for association was detected, suggesting that this gene will not have a substantial effect in determining susceptibility to RA in populations of Northern European descent.     

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.     

Along the road not taken: how many myosin heads act on a single actin filament at any instant in working muscle?

We reconsider the use of stiffness measurements to estimate N, the number of myosin heads acting (working at any instant to produce tension) on a single actin filament in vertebrate striated muscle, and give reasons for our rejection of numbers produced from such measurements. We go on to present a different approach to the problem, citing and extending a model bearing on the value of N which is derived from other physiological and biochemical data and which offers insight into the fundamental actin-myosin contractile event as an impulsive force. New experimental data accumulating over the past decade support this model, in which the myosin heads act sequentially along the actin filament (this is an example of Conformational Spread). In this model only a single myosin head acts on a single actin filament to produce an impulse at any given instant in normally-contracting muscle, either in the isometric or the isotonic mode, so N?=?1. However, extra impulses occur within the same time frame after quick release of length or tension. The predictions of this sequential model are in striking agreement with a large body of recent detailed biophysical and biochemical evidence. We suggest that this warrants further in-depth experimental work, specifically to explore and test the sequential model and its implications.     

Floral synorganization and its influence on mechanical isolation and autogamy in Marantaceae

The flowers of Marantaceae (～ 550 species) exhibit a highly derived pollination mechanism within Zingiberales, with a rapid and irreversible style movement based on a close synorganization of different floral parts. Given the complexity of the structure, we assume that little variation is possible if functionality is to be maintained. To test this, we investigated how much floral diversity exists in the clade and whether this diversity potentially influences the breeding system and placement of pollen on the pollinator. Flowers of 66 species covering the five major phylogenetic clades of the family were analysed. All species are similar in their basic flower construction: the fleshy staminode forms the tunnel‐shaped roof of the flower and narrows the tube with stiff swellings, and the hooded staminode holds the style under tension and narrows the flower entrance with its trigger appendage. Despite morphological diversity of the pollination apparatus, functionality is maintained by coordinated variation of the fleshy and hooded staminodes. Autogamy is usually avoided by herkogamy. However, in a few exceptions, subtle morphological changes alter the breeding system from allogamy to autogamy. Variable floral proportions allow for differential pollen deposition potentially causing mechanical isolation between sister taxa. This study clearly illustrates that structural variation is not only present in the highly synorganized flowers of Marantaceae, but that it also creates potentially new options for evolutionary diversification. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 300–322.     

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.