We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hsIVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome (3 subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coi expressing heat-shock Proteasomes are multicatalytic proteolytic complexes present in both the nucleus and cytosol of eukaryotic cells (1). The 26S form of the proteasome catalyzes the degradation of ubiquitinconjugated proteins (2-5), and thus it plays a key role in many cellular processes, including progression through the cell cycle (6, 7), removal of abnormal proteins, and antigen presentation (8). The proteolytic core of the 26S complex is the 20S (700 kDa) proteasome particle, which consists of four sevenmembered rings. The subunits of the 20S proteasome fall into two families (9, 10): the a-type forms the two outer rings, and P-type, which contain the active sites, forms the two inner rings of the complex.Proteasomes were thought to exist exclusively in eukaryotes and certain archaebacteria (11). However, 20S proteasomes were recently discovered in the actinomycete Rhodococcus (12), and in the Escherichia coli genome sequencing project, a novel heat-shock locus (hslVU) was discovered that encodes a 19-kDa protein (HslV) (13), whose sequence is similar to 3-type proteasome subunits. This discovery of proteasome-related genes was surprising, because several groups had failed to observe a structure in E. coli resembling the proteasome or proteins resembling ubiquitin. The hslV gene is cotranscribed with the adjacent hslU gene, which codes for a 50-kDa protein containing one ATP/GTP binding motif (13 For the expression of glutathione S-transferase (GST)-fusion proteins, the hslVand hslU genes were PCR amplified separately using A phage 18-126 DNA bearing the hslVU operon, kindly provided by F. Blattner (University of Wisconsin-Madison), and cloned into the vector pGEX-2T (Pharmacia). Vector pV106 (GST-HslV) and pU206 (GST-HslU) were electroporated into E. coli C600 cells. GST-fusion proteins were purified from strains C106 and C206 using the GST Purification Module (Pharmacia). To obtain antibodies, purified GST-HslV and GST-HslU proteins were injected into rabbits. Polyclonal anti-HslV and antiHslU antibodies were then affinity purified using the GST-fusions as ligands, and depleted of anti-GST antibodies using a GST column.Abbreviations: hsl, heat-shock locus; GST, glutathone S-transferase. tTo whom reprint requests should be addressed.5808

Extracorporeal shock waves (ESW) have recently been used in resolving tendinitis. However, mechanisms by which ESW promote tendon repair is not fully understood. In this study, we reported that an optimal ESW treatment promoted healing of Achilles tendintis by inducing TGF-PI and IGF-I. Rats with the collagenease-induced Achilles tendinitis were given a single ESW treatment (0.16 mJ/mm' energy flux density) with 0, 200, 500 and 1000 impulses. Achilles tendons were subjected to biomechanical (load to failure and stiffness), biochemical properties (DNA, glycosaminoglycan and hydroxyproline content) and histological assessment. ESW with 200 impulses restored biomechanical and biochemical characteristics of healing tendons 12 weeks after treatment. However, ESW treatments with 500 and 1000 impulses elicited inhibitory effects on tendinitis repair. Histological observation demonstrated that ESW treatment resolved edema, swelling, and inflammatory cell infiltration in injured tendons. Lesion site underwent intensive tenocyte proliferation, neovascularization and progressive tendon tissue regeneration. Tenocytes at the hypertrophied cellular tissue and newly developed tendon tissue expressed strong proliferating cell nuclear antigen (PCNA) after ESW treatment, suggesting that physical ESW could increase the mitogenic responses of tendons. Moreover, the proliferation of tenocytes adjunct to hypertrophied cell aggregate and newly formed tendon tissue coincided with intensive TGF-PI and IGF-I expression. Increasing TGF-Dl expression was noted in the early stage of tendon repair, and elevated IGF-I expression was persisted throughout the healing period. Together, low-energy shock wave effectively promoted tendon healing. TGF-PI and IGF-I played important roles in mediating ESW-stimulated cell proliferation and tissue regeneration of tendon.

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Summary Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ, and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.