Induced hypothyroidism alters articular cartilage in skeletally immature miniature swine.

PURPOSE/AIM: Thyroid hormone has been implicated in the normal growth and development of articular cartilage; however, its effect on a disease state, such as hypothyroidism, is unknown. The purpose of this investigation was to compare normal articular cartilage from proximal femurs of immature miniature swine to proximal femurs from hypothyroid-induced immature miniature swine. MATERIALS AND METHODS: Two 11-week-old male Sinclair miniature swine were made hypothyroid by administration of 6-propyl-2-thiouracil (PTU) in their drinking water; two control animals did not receive PTU. At 25 weeks of age, the animals were euthanized and their proximal femurs were fixed and decalcified. Samples were sectioned and analyzed by histology to define extracellular matrix (ECM) structure, immunohistochemistry (IHC) to identify types II and X collagen, and histomorphometry to assess articular cartilage mean total and localized height and cell density. Statistics included nested mixed-effects ANOVA with p ≤ 0.05 considered statistically significant. RESULTS: Compared to controls, hypothyroid articular cartilage demonstrated statistically significant quantitative differences in mean tissue height, mean cell density and type II collagen localized zone height. Qualitative differences in ECM proteoglycans and overall collagen types were also found. Type X collagen was not detected in either hypothyroid or control articular cartilage specimens. CONCLUSIONS: Significant changes in articular cartilage structure in hypothyroid compared to control immature miniature swine suggest that thyroid hormone is critical in the growth and development of articular cartilage. CLINICAL SIGNIFICANCE: Understanding articular cartilage development in immature animal models may provide insight into healing or repair of degenerative human articular cartilage.

5'-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the Drosophila central nervous system.

Drosophila RNP-4F is a highly conserved protein from yeast to human and functions as a spliceosome assembly factor during pre-mRNA splicing. Two major developmentally regulated rnp-4f mRNA isoforms have been described during fly development, designated "long" and "short," differing by a 177-nt tract in the 5'-UTR. This region potentially folds into a single long stable stem-loop by pairing of intron 0 and part of exon 2. Since the coding potential for the two isoforms is identical, the interesting question arises as to the functional significance of this evolutionarily-conserved 5'-UTR feature. Here we describe the effects of wild-type and mutated stem-loop on modulation of rnp-4f gene expression in embryos using a GFP reporter assay. In this work, a new GFP expression vector designated pUAS-Neostinger was constructed. The UAS-GAL4 system was utilized to trigger GFP expression using tissue-specific promoter driver fly lines. Fluorescence microscopy visualization, Western blotting and real-time qRT-PÇR were used to study and quantify GFP reporter protein and mRNA levels. A significant increase in GFP reporter protein expression due to presence of the wild-type stem-loop sequence/structure was unexpectedly observed with no concomitant increase in GFP reporter mRNA levels, showing that the 177-nt region enhancement acts posttranscriptionally. The effects of potential cis-acting elements within the stem-loop were evaluated using the reporter assay in two mutant constructs. Results of GFP reporter over-expression show that RNP-4F translational regulation is highly sensitive in the developing fly central nervous system. The potential molecular mechanism behind the observed translational enhancement is discussed.