Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

<p>Abstract</p> <p>Background</p> <p>Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release <it>ex vivo </it>of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors.</p> <p>Findings</p> <p>Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFα). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich ³⁴²FFGVG-G2 ELISA, (2) competition NITEGE³⁷³ELISA (3) sandwich G1-NITEGE³⁷³ELISA (4) competition ³⁷⁴ARGSV ELISA, and (5) sandwich ³⁷⁴ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE³⁷³neo-epitopes are delayed compared to the corresponding ³⁷⁴ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only.</p> <p>Conclusion</p> <p>Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.</p

Cell Suspension Culture-Mediated Incorporation of the Rice Bel Gene into Transgenic Cotton

Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 µmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops

Heterozygous Ldlr-Deficient Hamster as a Model to Evaluate the Efficacy of PCSK9 Antibody in Hyperlipidemia and Atherosclerosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis and atherogenesis. However, there are only limited rodent models, with a functional low-density lipoprotein receptor (LDLR) pathway and cholesteryl ester transfer protein (CETP) to evaluate the drug candidates targeting the PCSK9/LDLR pathway, that are translatable to humans. Here, by using our recently generated LDLR heterozygote (Ldlr+/&minus;) hamster model with functional LDLR pathway and CETP function, we seek to evaluate the effect of a PCSK9 antibody, evolocumab, on dyslipidemia and atherosclerosis compared with ezetimibe, an effective inhibitor of cholesterol absorption, as a positive therapeutic control. We show that the plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) were significantly increased in Ldlr+/&minus; hamsters fed a high-fat high-cholesterol (HFHC) diet; therefore, areas of atherosclerotic lesion in the aorta were obviously increased and positively correlated with plasma LDL-C and TC. Circulating free PCSK9 was downregulated by the HFHC diet and was undetectable in the evolocumab treated group, as expected. Most importantly, either evolocumab or ezetimibe treatment prevented HFHC diet-induced hyperlipidemia and subsequent atherosclerotic plaque formation. The results indicate that Ldlr+/&minus; hamsters fed an HFHC diet represent an ideal rodent model to evaluate drug candidates that affect LDLR pathways

Transgenic cotton plants in pots, greenhouse and experimental plot.

<p>A. Transgenic plants grafted onto rootstock of Coker312 showing resistance to bentazon and rootstock showing sensitivity to bentazon (Arrow shows wilting 2 days after spraying with Bentazon), B. T0 transgenic cotton plant in greenhouse, C. T1 generation plants showing resistance to Bentazon, and some that are sensitive to Bentazon. D. T2 plants resistant to Bentazon and non-transgenic Coker 312 plants sensitive to Bentazon in the experimental plot.</p

Scheme for <i>Agrobacterium</i>-mediated transformation of suspension cultures and plant regeneration in upland cotton.

<p>Scheme for <i>Agrobacterium</i>-mediated transformation of suspension cultures and plant regeneration in upland cotton.</p

PCR and Southern blot analysis of transgenic cotton.

<p>A. PCR-based verification of stable insertion of the <i>npt II</i> gene into the genomic DNA of cotton using an <i>npt II -</i>specific probe; and B, of the <i>bel</i> gene with a <i>bel-</i>specific probe, C. Southern blot of T1 plants, Lane 1 shows positive control plasmid DNA, D. <i>Bel</i> expression analysis of RT-PCR of six plants of individual T2 and T3 plants, using <i>actin</i> as internal control. (Lane 1 showing Coker 312 as a negative control, Lane 2–7 showing T2 plants, Lane 8–13 showing T3 plants generated from T2 plants from plants in Lanes 2–7; <i>Actin</i> gene in cotton as reference gene).</p

The percentage of transformed colonies resistant to Bentazon and Kanamycin according to duration of suspension culture.

<p>R-colonies represent the number of colonies resistant to Bentazon and Kanamycin from the infected suspension cultures in one bottle, the transformation was repeated 3 times with different suspension cultures (1, 15, 30, 45 and 60 day-old); – represents callus colonies polluted by <i>Agrobacterium</i>; data were counted from each bottle.</p

Formation of transformed callus resistant to Bentazon and Kanamycin and transgenic plant regeneration via somatic embryogenesis.

<p>A. Infected suspension cultures on solid medium, B. Cell masses browning and new callus formation, C. New somatic embryogenic callus formation, D–E. Callus proliferation and somatic embryogenesis from different colonies, F–H. Somatic embryo formation, maturity, I. Cotton plant regeneration on medium supplemented with Kanamycin and Bentazon.</p