Christian leader as overcomer through principled story

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Human osteoarthritis synovium contains an alternatively spliced transcript of ADAMTS4

Purpose: The characterization of an alternatively spliced transcript of the ADAMTS4 aggrecanase. Methods: In human OA synovial cell cultures, RT-PCR was performed using oligonucleotide primers designed to amplify across the exon 8/9 region of human ADAMTS4. The PCR products were purified using a QIAquick purification kit (Qiagen) and sequenced using in house facilities. A pCEP4 (Invitrogen) mammalian expression vector containing ADAMTS4 plus a FLAG epitope was mutated using the QuikChange II site directed mutagenesis kit (Stratagene) to contain the ADAMTS4 splice variant plus a FLAG epitope. The recombinant proteins were purified from HEK293 transfected cells using Anti-FLAG M2 affinity gel (Sigma). Polyclonal antibodies were raised against synthetic peptides representing sequences within the C-terminal region of the splice variant of ADAMTS4 and the raised antibodies were characterized using the recombinant splice variant of ADAMTS4. The antibodies were used in immunohistochemical analysis of human osteoarthritic synovium. The proteolysis of aggrecan and other proteoglycans by the recombinant spice variant of ADAMTS4 was investigated. Results: The degradation of aggrecan is mainly mediated by the aggrecanases, of which ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) are the best known. We here characterize an alternative splice variant of ADAMTS4. RT-PCR performed as described above resulted in the amplification of normal ADAMTS4, and also a smaller product missing 161 base pairs from the 5’ end of exon 9, the result of alternative splicing in which exon 8 joins to a cryptic 3’ splice site within exon 9. The protein produced by this alternative splicing would lack the spacer domain and have a C-terminus lacking any homologies with the normal ADAMTS4 spacer domain. The alternatively spliced transcript of ADAMTS4 was found in cultured OA synovial cells and in freshly digested OA synovium, but not in human brain, cervix or lung, or in normal bovine synovium. The protein synthesized from this alternatively spliced transcript of ADAMTS4 would lose functions dependent on its spacer domain, like substrate and matrix binding, and inhibition through fibronectin. Removal of the spacer domain from ADAMTS4 has been reported to increase its ability to cleave aggrecan at the Glu373-Ala374 bond, and it may well be that the alternatively spliced transcript produces a protein that is secreted in a more active form. HEK293 cells transfected with a pCEP4 vector containing the cDNA sequence of the splice variant of ADAMTS4 produced the corresponding protein in both the pro and active form. This protein could be found in the media, but mostly associated with the cells, as confirmed using antibodies specific for the splice variant that were produced using synthetic peptides. Immunohistochemical analysis of osteoarthritic synovium using these antibodies showed staining of cells within the synovium. Proteins purified by immunoprecipitation by Anti-FLAG M2 affinity gel from transfected and untransfected HEK293 cells were analysed using the ANASpec SensoLyte 520 Aggrecanase I assay kit. The splice variant had aggrecanase activity comparable to a commercially available ADAMTS4. The splice variant cleaved aggrecan at the G1u373-A1a374 site, as assessed by the neoepitope monoclonal antibody BC3, with activity comparable to ADAMTS4. Conclusions: ADAMTS4 is regulated at multiple levels through control of gene expression, mRNA splicing and protein processing, as well as the expression of naturally occurring inhibitors. We here describe the characteristics of the first known splice variant of ADAMTS4. This alternative splice transcript of ADAMTS4 is expressed as a protein in vivo and can be found in the synovium. It can be speculated that the changes in the C-terminal domain of the protein resulting from this alternatively spliced transcript would have changes in its substrate specificity. The protein produced by the alternative spliced transcript of ADAMTS4 has aggrecanase activity, and the release of low levels of this fully active variant of ADAMTS4 might be a factor in the slow process of superficial zone aggrecan loss in osteoarthritis

Management of obesity and cardiometabolic risk - role of phentermine/extended release topiramate

The US Food and Drug Administration (FDA) recently approved lorcaserin and the combination of phentermine and extended release topiramate (phentermine/topiramate ER) for the treatment of obesity in conjunction with a lifestyle intervention, expanding the therapeutic options for long-term obesity pharmacotherapy, which was previously limited to orlistat. Combination phentermine/topiramate ER is associated with greater weight loss compared to its constituent monotherapy, with a more favorable adverse effect profile. Phentermine/topiramate ER also appears to have beneficial effects on cardiometabolic risk, although longer-term cardiovascular safety data are required. While there are no head-to-head studies among the currently available obesity pharmacotherapy agents, phentermine/topiramate ER appears to have a superior weight loss profile. This review will discuss the epidemiology, natural history, and cardiometabolic risk associated with obesity, provide an overview on current obesity pharmacotherapy, and summarize the recent clinical efficacy and safety data underpinning the FDA's approval of both phentermine/topiramate ER and lorcaserin as pharmacotherapy for a long-term obesity intervention

Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan

Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan

Egg Consumption and Human Cardio-Metabolic Health in People with and without Diabetes

The guidelines for dietary cholesterol and/or egg intake for both the general population and those at higher risk of cardiovascular disease (for example, people with type 2 diabetes mellitus (T2DM)) differ between countries, and even for different specialist societies in a country. The disparity between these guidelines is at least in part related to the conflicting evidence as to the effects of eggs in the general population and in those with T2DM. This review addresses the effect of eggs on cardiovascular disease (CVD) risk from both epidemiological research and controlled prospective studies, in people with and without cardio-metabolic disease. It also examines the nutritional qualities of eggs and whether they may offer protection against chronic disease. The evidence suggests that a diet including more eggs than is recommended (at least in some countries) may be used safely as part of a healthy diet in both the general population and for those at high risk of cardiovascular disease, those with established coronary heart disease, and those with T2DM. In conclusion, an approach focused on a person’s entire dietary intake as opposed to specific foods or nutrients should be the heart of population nutrition guidelines