K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/ BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca 2 þ homeostasis. Neither cytosolic chaperones nor Cdc48p/ Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where b is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysinefree K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.

Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

This approach may be of value to study the effects of potential therapeutic genes upon cartilage repair in a clinically relevant setting.

Osteoarthritis (OA) is a chronic disorder of the diarthrodial joints, mostly characterized by gradual deterioration of the articular cartilage. This disease still has no effective treatment. An emerging strategy for treating OA is based on molecular concepts using growth factors, transcription factors, and signaling molecules in light of their effects on the restoration of cartilage integrity. Recent studies have demonstrated that overexpression of such candidate molecules using direct gene transfer or ex vivo protocols is capable of stimulating cell proliferation and matrix synthesis in normal human and OA cartilage explants in vitro as well as in animal models in vivo. As a result, the structure of the articular cartilage can be improved. More insights into the pathophysiology of human OA and further studies in animal models are needed, however, to facilitate clinical translation of these molecular approaches. In conclusion, recent experimental findings permit cautious optimism, holding promise for treating human OA in the future.

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