Altered specificity of a AAA+ protease.

ClpXP, an ATP-dependent protease, degrades hundreds of different intracellular proteins. ClpX chooses substrates by binding peptide tags, typically displayed at the N or C terminus of the protein to be degraded. Here, we identify a ClpX mutant that displays a 300-fold change in substrate specificity, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. The altered-specificity mutation occurs within "RKH" loops, which surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. These results support a major role for the RKH loops in substrate recognition and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.

Patient and physician-assessed shoulder function after arthroplasty.

BACKGROUND: We found no information in the literature regarding the relationship between patient and physician-derived outcome assessments with a shoulder questionnaire. In this study, we examined a group of patients who were assessed with patient and physician-administered questionnaires following shoulder arthroplasty. METHODS: From August 2003 to February 2004, sixty-seven consecutive patients who had been followed for a minimum of six months after shoulder arthroplasty were evaluated with a self-administered and an identical physician-directed shoulder questionnaire that assessed clinical and functional outcomes at the time of routine follow-up. An assessment of the agreement between physicians and patients as well as the factors that affected agreement was performed. RESULTS: The intraclass correlation indicated almost perfect physician-patient agreement (>0.80) on items related to overall pain, pain at night, pain with activity, stability, and active elevation and substantial agreement (intraclass correlation, 0.66 and 0.69) between the physician and patient assessments of pain without activity and strength. While the differences were small, on the average physician ratings for pain were lower (indicating less pain) than patient ratings for pain, physicians rated stability and strength as being closer to normal, and they reported less active elevation. There was substantial agreement between the physician and patient assessments of outcome with the modified Neer system (intraclass correlation = 0.75), with 87% agreement if excellent and satisfactory outcomes were combined. CONCLUSIONS: A patient-derived questionnaire can provide a high level of agreement with surgeon assessments of outcome following shoulder surgery. Patient-administered methods should continue to be evaluated as a means of assessment of these patients.

Motor nerve palsy following primary total hip arthroplasty.

BACKGROUND: Nerve palsy is a potentially devastating complication following total hip arthroplasty. The purpose of this study was to retrospectively identify risk factors for, and the prognosis associated with, a motor nerve palsy following primary total hip arthroplasty. METHODS: Between 1970 and 2000, 27,004 primary total hip arthroplasties were performed at our institution. Forty-seven patients (0.17%) with postoperative motor nerve dysfunction were identified by a review of the complications log of a total joint database. The medical record of each patient provided the data for this study. The average age of the patients was fifty-seven years at the time of surgery. The patients had serial clinical examinations for a minimum of two years, or until neurologic recovery or death. The nerve palsies were classified as complete or incomplete, and only patients with objective motor weakness were included in the study. The limb lengths were measured on preoperative and postoperative radiographs, and those data were then compared with the limb lengths in a matched cohort of patients who had not sustained a nerve injury after a primary total hip arthroplasty. The extent of neurologic recovery, the need for braces or walking aids, and the use of medications for neurogenic pain were evaluated. RESULTS: There were twenty-nine complete motor nerve palsies (sixteen peroneal, eleven sciatic, and two femoral) and eighteen incomplete motor nerve palsies (fourteen peroneal, three sciatic, and one femoral). A preoperative diagnosis of developmental dysplasia of the hip (p = 0.0004) or posttraumatic arthritis (p = 0.01), the use of a posterior approach (p = 0.032), lengthening of the extremity (p < 0.01), and cementless femoral fixation (p = 0.03) were associated with a significantly increased odds ratio for the development of a postoperative motor nerve palsy. Of the twenty-eight patients with a complete palsy who were available for follow-up, only ten (36%) had complete recovery of motor strength, which took an average of 21.1 months. Seven of the eighteen patients with an incomplete palsy fully recovered their preoperative strength. Twenty-one patients required walking aids, and fifteen required permanent use of an ankle-foot orthosis. Five patients required daily medication for chronic neurogenic pain. CONCLUSIONS: Motor nerve palsy is uncommon following primary total hip arthroplasty. A preoperative diagnosis of developmental dysplasia of the hip or posttraumatic arthritis, the use of a posterior approach, lengthening of the extremity, and use of an uncemented femoral implant increased the odds ratio of sustaining a motor nerve palsy. The majority of the motor nerve deficits in our series, whether complete or incomplete, did not fully resolve.

Manipulation for frozen shoulder: long-term results.

Although much has been published regarding shoulder manipulation under anesthesia for the treatment of frozen shoulder, there are no reported long-term results. In 25 patients (26 shoulders) in whom nonoperative treatment for idiopathic frozen shoulder had failed, we performed manipulation under anesthesia. All had had physical therapy for a mean of 6.2 months. Follow-up was by examination until the end of active treatment. Longer-term follow-up was obtained in 18 patients (19 shoulders) by questionnaire and averaged 15 years (range, 8.1 to 20.6 years). There were significant improvements in forward elevation from a mean of 104 degrees before manipulation (range, 70 degrees to 140 degrees ) to 168 degrees (range, 90 degrees to 180 degrees ) and in external rotation from 23 degrees (range, -5 degrees to 70 degrees ) to 67 degrees (range, 0 degrees to 90 degrees ). There were 16 shoulders with no pain or slight pain and 3 with occasional moderate or severe pain. There were no fractures, dislocations, or other complications. Of the 19 shoulders, 18 required no further surgery. At long-term follow-up, the mean Simple Shoulder Test score was 9.5 out of 12 and the mean American Shoulder and Elbow Surgeons score was 80 out of 100. Treatment of idiopathic frozen shoulder by manipulation under anesthesia leads to sustained improvement in shoulder motion and function at a mean of 15 years after the procedure.

Autogenous femoral head bone grafts for acetabular deficiency in total-hip arthroplasty for developmental dysplasia of the hip: long-term effect on pelvic bone stock.

Twenty-eight primary total-hip arthroplasties in 23 patients performed with autogenous femoral head bone graft augmentation for developmental hip dysplasia were retrospectively reviewed at 8 to 15 years. Five sockets were revised for different reasons. At revisions, 3 grafts were healed, the other 2 had substantial resorption. Immediately postoperatively, the mean medial to lateral graft thickness at the level of the superior border of the socket was 33 mm. Radiographs a mean of 4.8 years after operation demonstrated this thickness decreased by a mean of 1 mm (range, 0-10 mm). At 11 years (range, 8-15 years), radiographs in unrevised hips showed no cases of substantial further graft resorption.

Cytoplasmic degradation of ssrA-tagged proteins.

Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.

Synthesis of 9-acridinyl sulfur derivatives: sulfides, sulfoxides and sulfones. Comparison of their activity on tumour cells.

The synthesis of several acridine thioethers is described. These compounds were oxidized to give new sulfoxides and sulfones. Among 23 compounds prepared, 19 were tested in vitro against the human cancer cell lines panel of NCI screening. Activity is increased 5-10 times from sulfides to sulfoxides. Among substituted groups in the side chain, sulfur mustard, epoxy sulfide and sulfoxide displayed the most interesting activity.

A kinesin switch I arginine to lysine mutation rescues microtubule function.

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.

Acute compartment syndrome of the leg following diagnostic electromyography.

Acute compartment syndrome of the lower extremity is typically associated with some form of trauma, either fracture or blunt injury. Accurate diagnosis and urgent treatment of this condition is required for preservation of the viability of the limb. We report the case of a 73-year-old man who developed an acute compartment syndrome of the leg after diagnostic electromyography. Potential causes and treatment are discussed.

The role of ATP hydrolysis for kinesin processivity.

Conventional kinesin is a highly processive, plus-end-directed microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our pre-steady-state kinetic analysis of a kinesin switch I mutant at Arg(210) (NXXSSRSH, residues 205-212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.