Aip1p interacts with cofilin to disassemble actin filaments.

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.

Effect of synovitis and corticosteroids on transcription of cartilage matrix proteins.

OBJECTIVE: To determine whether steady-state levels of type-II procollagen, aggrecan core protein, or fibronectin mRNA in articular chondrocytes are altered by synovitis or administration of methylprednisolone acetate (MPA). SAMPLE POPULATION: Articular cartilage specimens collected from 10 ponies, 2.5 to 3.5 years old and 200 to 300 kg. PROCEDURE: 4 experimental groups were compared, using the cartilage specimens: control, MPA-treated, lipopolysaccharide-induced synovitis, and lipopolysaccharide-induced synovitis with MPA treatment. RNA was isolated from articular cartilage and compared by northern blot analysis, using equine-specific cDNA probes. RESULTS: Synovitis increased steady-state levels of type-II procollagen mRNA fivefold and of aggrecan mRNA twofold. Administration of a single intra-articular injection of MPA (0.1 mg/kg of body weight) decreased type-II procollagen transcripts in normal cartilage sixfold, without significant effect on aggrecan or total fibronectin mRNA values. MPA treatment of inflamed joints decreased type-II procollagen and aggrecan mRNA to levels that were not significantly different from those in untreated control specimens. CONCLUSIONS: Articular chondrocytes increase type-II procollagen and aggrecan synthesis in response to synovitis. MPA alters chondrocyte function in normal and inflamed cartilage, suggesting that potential changes in cartilage matrix protein synthesis should be considered when evaluating the therapeutic value of intra-articular administration of corticosteroids. CLINICAL RELEVANCE: Knowledge of how synovitis and corticosteroids (independently and in combination) affect synthesis of cartilage matrix proteins is relevant to understanding pathogenesis of traumatic osteoarthritis and improving therapeutic strategies.

Expression and bioactivity of recombinant canine erythropoietin.

OBJECTIVE: To produce recombinant canine erythropoietin (rcEPO) and compare its biological activity with that of recombinant human EPO (rhEPO). ANIMALS: C57BL/6J mice. PROCEDURE: The gene encoding cEPO was isolated from a genomic library and subcloned into an eucaryotic expression vector. Production of rcEPO was achieved by stable transfection of the expression construct into Chinese hamster ovary cells. Biological activity was evaluated in vitro by analyzing the mitogenic activity of rcEPO on murine erythroid progenitor cells. In vivo bioactivity was assessed in mice by measuring the ability of rcEPO to increase blood reticulocyte counts. RESULTS: Size and glycosylation of rcEPO expressed in Chinese hamster ovary cells were similar to values for commercial rhEPO. Canine and human EPO stimulated proliferation of murine erythroid progenitor cells in vitro and murine reticulocytosis in vivo in a dose-dependent manner. CONCLUSIONS: Comparable biological activity was observed for rcEPO and rhEPO in the 2 murine-based assay systems studied. By avoiding interspecies variation in protein structure and the resulting potential for immunogenicity, rcEPO should represent a better option than rhEPO for treatment of dogs with erythropoietin-dependent anemia. CLINICAL RELEVANCE: Therapeutic use of rhEPO in companion animals is limited by its immunogenicity and the resulting potential to induce pure red cell aplasia. Development and availability of species-specific EPO preparations should avoid this problem.

The cartilage-specific (V+C)- fibronectin isoform exists primarily in homodimeric and monomeric configurations.

Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)(-) splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)(-) isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)(-) fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)(-) monomers were also present. Heterodimers of (V+C)(-) with V(+),C(+) (V120) isoforms were not detected. Heterodimers of (V+C)(-) with V(-),C(+) (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)(-) fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.