Differential effects of interleukin-1 and transforming growth factor β on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers

Small proteoglycans (PGs) are supposed to play great roles in the assembly of cartilage matrix but the influence of cytokines and growth factors on their synthesis by articular chondrocytes is largely unknown. We investigated whether EL-1 and TGF1 influence the production of small leucine-rich proteoglycans by chondrocytes cultured in a three-dimensional gel, as compared to the common monolayer system.Rabbit articular chondrocytes were cultured in alginate beads for 14 days or as monolayers for 7 days. The effect of 2 ng/ml IIL-1 or TGF1 during the last two days in culture was determined, after [³⁵S]methionine labeling over the last 24 h. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment of alginate beads whereas medium and cell-layer fractions were isolated from monolayer cultures. Total newly synthesized PGs were first isolated by anion-exchange chromatography and the small PGs were further separated from aggrecans by gel-filtration (Sepharose CL-4B) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).Addition of TGF1 resulted in an overall rise in neosynthesized small PG content in both culture systems. However, TGF1 significantly increased to the same extent the percentage of small PGs laid down in the cell-associated and the further-removed matrix compartments of the beacls culture (+00%) whereas it auirnted the content of small PGs in the medium (+40%) and reduced that of the cell fraction (+35%) in the monolayer culture. By adding IL-1, the amount of total newly synthesized small PGs was decreased in monolayers while it increased in alginate beads. IL-1 was also shown to change the relative distribution of these molecules in the monolayer system in contrast to the alginate beads culture where the proportions were not significantly altered. Electrophoretic analyes of the ³⁵S-labeled small PGs-containing fractions confirmed these effects at the level of the 45-50 kDa-related core proteins.This study demonstrates that TGF and IL-1 differently influence small PG synthesis of rabbit articular chondrocytes depending on whether they are cultured in alginate beads or in monolayers. Moreover, the regulation of small PG expression appears to be different from that of high-molecular weight aggrecans. As these small molecules are playing major roles in matrix assembly and growth factor regulation, the data may have great relevance to the pathogenesis of osteoarthritis and repair of articular cartilage lesions.     

Evidence of diversity and recombination in <Emphasis Type=Italic>Arsenophonus</Emphasis> symbionts of the <Emphasis Type=Italic>Bemisia tabaci</Emphasis>species complex

BACKGROUND: Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology. RESULTS: Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers. CONCLUSIONS: This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.     

Chronic bee paralysis: A disease and a virus like no other?

Chronic bee paralysis which was called Paralysis is a rather unusual disease caused by a rather unusual virus. In this review, we explore current knowledge of the disease and its etiological agent. Paralysis is the only common viral disease of adult bees whose symptoms include both behavioural and physiological modifications: trembling and hair loss. The disease often affects the strong colonies of an apiary and thousands of dead individuals are then observed in front of the hives. Two sets of symptoms have traditionally been described in the existing literature, but nowadays we can define a general syndrome.The morphology of the Chronic bee paralysis virus (CBPV) particles and the multipartite organisation of the RNA genome are exceptional, as most honey bee viruses are picorna-like viruses belonging to the Iflavirus and Cripavirus genera with symmetric particles and monopartite positive, single-strand RNA genomes. CBPV is currently classified as an RNA virus but is not included in any family or genus. Although it shares several characteristics with viruses in the Nodaviridae and Tombusviridae families, it differs from previously known viruses according to the various demarcation criteria defined by the International Committee on Taxonomy of Viruses (ICTV). Thus, it should be considered as the type species of a new group of positive-strand RNA viruses.The recent sequencing of the complete CBPV genome has opened the way for phylogenetic studies and development of new molecular tools able to detect variable isolates and to quantify genomic loads. This article considers the results of such recent detection tests but also previous studies including: (i) the distribution of CBPV infection within the bees and the hive, (ii) the way the virus spreads and its persistence in the colony environment, and (iii) geographical and seasonal distribution and impact of CBPV infections.     

Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.     

Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo

BACKGROUND: Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. METHODS: Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation. RESULTS: In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo. CONCLUSIONS: The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects.     

ATP synthesis and proton handling in muscle during short periods of exercise and subsequent recovery.

We used (31)P-magnetic resonance spectroscopy to study proton buffering in finger flexor muscles of eight healthy men (25-45 yr), during brief (18-s) voluntary finger flexion exercise (0.67-Hz contraction at 10% maximum voluntary contraction; 50/50 duty cycle) and 180-s recovery. Phosphocreatine (PCr) concentration fell 19 +/- 2% during exercise and then recovered with half time = 0.24 +/- 0.01 min. Cell pH rose by 0.058 +/- 0.003 units during exercise as a result of H(+) consumption by PCr splitting, which (assuming no lactate production or H(+) efflux) implies a plausible non-P(i) buffer capacity of 20 +/- 3 mmol. l intracellular water(-1). pH unit(-1). There was thus no evidence of significant glycogenolysis to lactate during exercise. Analysis of PCr kinetics as a classic linear response suggests that oxidative ATP synthesis reached 48 +/- 2% of ATP demand by the end of exercise; the rest was met by PCr splitting. Postexercise pH recovery was faster than predicted, suggesting "excess proton" production, with a peak value of 0.6 +/- 0.2 mmol/l intracellular water at 0.45 min of recovery, which might be due to, e.g., proton influx driven by cellular alkalinization, or a small glycolytic contribution to PCr resynthesis in recovery.