Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.     

Cell adhesion molecule in the hexactinellid Aphrocallistes vastus

Abstract. The Hexactinellida sponge Aphrocallistes vastus contains a soluble aggregation factor (AF) whose purification has been described in this communication. It is characterized by a S°_20.w value of 37 and a buoyant density of 1.45 g/cm³. The AF is a glycoporteinaceous particle composed of three major protein species; no core structure could be visualized. In the presence of Ca²⁺, the AF causes secondary aggregation of single cells. The aggregation process is temperature, pH, and ionic strength independent within a broad range. Evidence is presented indicating that two (or more) AF molecules are required for the establishment of a stable cell: cell interaction. In contrast to the AFs from demosponges, the hexactinellid AF functions species-unspecifically.     

Predicting Effects of Water Regime Changes on Waterbirds: Insights from Staging Swans

Predicting the environmental impact of a proposed development is notoriously difficult, especially when future conditions fall outside the current range of conditions. Individual-based approaches have been developed and applied to predict the impact of environmental changes on wintering and staging coastal bird populations. How many birds make use of staging sites is mostly determined by food availability and accessibility, which in the case of many waterbirds in turn is affected by water level. Many water systems are regulated and water levels are maintained at target levels, set by management authorities. We used an individual-based modelling framework (MORPH) to analyse how different target water levels affect the number of migratory Bewick’s swans Cygnus columbianus bewickii staging at a shallow freshwater lake (Lauwersmeer, the Netherlands) in autumn. As an emerging property of the model, we found strong non-linear responses of swan usage to changes in water level, with a sudden drop in peak numbers as well as bird-days with a 0.20 m rise above the current target water level. Such strong non-linear responses are probably common and should be taken into account in environmental impact assessments.     

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

Mycolactone toxin induces an inflammatory response by targeting the IL-1β pathway: Mechanistic insight into Buruli ulcer pathophysiology

Mycolactone, a lipid-like toxin, is the major virulence factor of Mycobacterium ulcerans, the etiological agent of Buruli ulcer. Its involvement in lesion development has been widely described in early stages of the disease, through its cytotoxic and immunosuppressive activities, but less is known about later stages. Here, we revisit the role of mycolactone in disease outcome and provide the first demonstration of the pro-inflammatory potential of this toxin. We found that the mycolactone-containing mycobacterial extracellular vesicles produced by M. ulcerans induced the production of IL-1β, a potent pro-inflammatory cytokine, in a TLR2-dependent manner, targeting NLRP3/1 inflammasomes. We show our data to be relevant in a physiological context. The in vivo injection of these mycolactone-containing vesicles induced a strong local inflammatory response and tissue damage, which were prevented by corticosteroids. Finally, several soluble pro-inflammatory factors, including IL-1β, were detected in infected tissues from mice and Buruli ulcer patients. Our results revisit Buruli ulcer pathophysiology by providing new insight, thus paving the way for the development of new therapeutic strategies taking the pro-inflammatory potential of mycolactone into account.     

Correlation of adenosine receptor affinities and cardiovascular activity

Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [3H]NECA) were compared to their EC25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity (r2 = 0.71, p less than 0.0001) but not with A2 binding affinity (r2 = 0.007, n.s.); conversely, coronary flow (an A2 response) correlated with A2 binding affinity (r2 = 0.83, p less than 0.0001) but not with A1 binding affinity (r2 = 0.05, n.s.). These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses.     

Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells

A ribonuclease which was previously shown to be located in isolated vacuoles from suspension-cultured cells of tomato (Lycopersicon esculentum L.; Abel and Glund 1986, Physiol. Plant. 66, 79–86) has been purified to near homogeneity. Purification was up to 55000-fold with a yield of about 20%. The vacuolar origin of the protein was evidenced by comparing its electrophoretic mobility, isoelectric point, pH-optimum for activity and other properties with that of the RNA-degrading activity present in isolated vacuoles. The molecular weight of the native single polypeptide chain was estimated at 17500 and 20300 by gel filtration and sedimentation analysis, respectively. The enzyme hydrolyzed only single-stranded RNA with a mode of action that was endonucleolytic. The vacuolar ribonuclease had no requirement for divalent metal ions, and did not exhibit phosphomonoesterase (EC 3.1.3.1; EC 3.1.3.2) and phosphodiesterase (EC 3.1.15.1; EC 3.1.16.1) activity. The specificity of the enzyme has been studied by using homopolyribonucleotides as substrates. The end-products obtained were the respective nucleoside 2:3-cyclic monophosphates and, to minor extents, the corresponding nucleoside 3(2)-monophosphates. According to these observations, the vacuolar ribonuclease from tomato can be classified as ribonuclease I (EC 3.1.27.1).Abbreviations DEAE diethylaminoethyl - RNase ribonuclease - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

High-affinity uptake of γ-[3H]aminobutyric acid by isolated mouse oligodendrocytes in culture

Oligodendrocytes were isolated from mixed glial cultures of neonatal mouse forebrain and further grown in serum-free hormone supplemented culture medium. Cell populations were identified by indirect immunofluorescence using a range of specific antibodies, revealing a predominantly immature population of oligodendrocytes, the majority expressing the myelin glycolipids galactocerebroside and sulfatide on their plasma membrane. Astroglial contamination was found to be minimal. Simultaneous autoradiography and immunofluorescence demonstrated the presence of a transport system for the major inhibitory neurotransmitter GABA in the oligodendrocytes. The transport system was found to be energy, sodium and temperature dependent. Kinetic analysis revealed a high affinity system, with aK _m of 6.27 M and aV _max of 0.714 nmol/min/mg protein, which is comparable to that found previously for CNS neurons and astrocytes.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.