S-nitrosylation and S-glutathiolation of protein sulfhydryls by S-nitroso glutathione

The modification of reactive protein sulfhydryls by S-nitrosoglutathione and other NO donors has been studied by gel isoelectric focusing. S-nitrosylated, unmodified, and S-glutathiolated protein forms are differentiated by this method. With specific antibodies for the protein of interest, both S-nitrosylation and S-glutathiolation of the protein were analyzed in mixtures obtained as soluble tissue or cell extracts. The effect of S-nitrosoglutathione (GSNO) on purified phosphorylase b, on carbonic anhydrase III in an extract from rat liver, and on H-ras expressed in Escherichia coli was examined. When fresh GSNO reacted with pure phosphorylase b, only S-nitrosylated forms of the protein were observed. Likewise the NO donors, amyl nitrite, spermine NONOate, and diethylamine NONOate, all generated S-nitrosylated phosphorylase b. When crude mixtures of proteins from rat liver (containing carbonic anhydrase III) or from E. coli (containing an overexpressed form of H-ras) were exposed to fresh GSNO, both the S-nitrosylated and the S-glutathiolated forms of the proteins were observed. It is suggested that reactive intermediates from the breakdown of GSNO are responsible for the observed S-glutathiolation. These experiments show that both S-nitrosylated and S-glutathiolated forms of proteins may be generated by the addition of GSNO to mixtures containing proteins with reactive sulfhydryls. These protein modifications may exhibit metabolic consequences independent of the release of nitric oxide.     

The effect of exercise and rehabilitation on anterior-posterior knee displacements after anterior cruciate ligament autograft reconstruction

We studied the effect of rehabilitation strength training and return to activities on anterior-posterior knee displacements after patellar tendon autogenous anterior cruciate ligament reconstruction. A total of 938 measurements were sequentially collected for 142 patients with the KT-2000 arthrometer. Rehabilitation included immediate knee motion and early weightbearing, light sports at 6 months, and competitive sports at 8 months or later. At a minimum of 2 years after surgery, 121 patients (85%) had normal displacements (less than 3 mm of increase at 134 N), 14 (10%) had 3 to 5.5 mm of increase (partial function), and 7 (5%) had more than 5.5 mm of increase (failed). There was no association found between the initial onset of the abnormal displacements in the 21 knees and either the amount of time after surgery or the rehabilitation program. Six of the seven grafts that failed did so in the 1st postoperative year. Serial displacement measurements allow early detection of graft stretching and subsequent modification of rehabilitation or delay in return to strenuous activities. These measurements showed that the rehabilitation program used in this study was not itself injurious and resulted in an acceptable failure rate of 5%.     

Is it possible to predict limb viability in complex Gustilo IIIB and IIIC tibial fractures? A comparison of two predictive indices

The patient with severe lower limb trauma presents a management dilemma; whether to amputate primarily or to attempt limb salvage. In recent years, many predictive indices have been published which purport to identify limbs which are non-viable. We retrospectively applied two recently described indices, the Mangled Extremity Severity Score (MESS) and the Limb Salvage Index (LSI), to 54 limbs in 50 patients with either Gustilo IIIB or IIIC complex tibial fractures. There were 22 amputations (40.7 per cent) in the series. The mean MESS score in the limb salvage group was 3.8 (range 2-10), and the mean MESS score in the amputation group was 7.7 (range 4-13) (P < 0.0001). The mean LSI score in the limb salvage group was 3.6 (range 3-8), and the mean LSI score in the amputation group was 6.9 (P < 0.01). However, in the group with MESS scores > 7 (which recommends amputation), there were three limbs which were salvaged with acceptable functional outcome. Similarly, in those with LSI scores > 6 (which recommends amputation), there were seven limbs successfully salvaged. A MESS > 7 offered a greater relative risk of amputation (9.2) than a LSI score > 6 (5.3). We found both indices of use in predicting limb salvage and functional outcome. However, neither is sufficiently accurate to be considered absolutely reliable in clinical practice.     

Long-term intravesical oxybutynin chloride therapy in children with myelodysplasia

PURPOSE: We evaluated the clinical use of long-term intravesical oxybutynin chloride in the treatment of neurogenic bladder dysfunction in children with myelodysplasia who could not tolerate oral anticholinergics. MATERIALS AND METHODS: We retrospectively reviewed the records of all patients recommended for intravesical oxybutynin chloride therapy. A total of 12 girls and 18 boys 1 to 17 years old was recruited for study. Oxybutynin chloride (5 mg.) was instilled 2 times daily and pretreatment cystograms were compared to followup urodynamic studies. Duration of therapy was 2 to 26 months (mean 13, median 12). RESULTS: Mean total capacity plus or minus standard deviation increased from 209 +/- 103 to 282 +/- 148 ml. (p < 0.01), mean safe capacity increased from 157 +/- 105 to 234 +/- 147 ml. (p < 0.01) and mean age adjusted safe capacity increased from 76 +/- 36 to 115 +/- 62%. Of the 29 patients who were incontinent 3 (10%) achieved continence and 19 (65%) reported a decreased use of sanitary pads. None of the patients had systemic side effects related to intravesical treatment. CONCLUSIONS: We believe that intravesical oxybutynin chloride is a viable treatment option for patients with myelodysplasia in whom oral therapy fails.     

Ultrasound screening for deep venous thrombosis after total knee arthroplasty. 2-year reassessment

The efficacy of ultrasound compared with ascending venography for the detection of deep venous thrombosis immediately after total knee arthroplasty was assessed after a 2-year interval. One hundred thirty-seven patients were eligible for the study; however, 31 patients received only one of the screening methods and a color Doppler examination was inconclusive in six patients. Therefore, 100 patients had a Doppler examination and a venogram. Overall, the sensitivity of ultrasound was 85%, the specificity 97%, the positive predictive value 85%, the negative predictive value 97%, and the accuracy 95%. The sensitivity in the calf was 83%, in the popliteal vein 86%, and in the femoral vein 100%. Two years ago, the initial assessment of ultrasound for the detection of deep venous thrombosis after surgery in patients who had total joint arthroplasty revealed a 75% sensitivity, 99% specificity, 91% positive predictive value, 97% negative predictive value, and 97% accuracy. The sensitivity in the calf was 83%; the sensitivity in the popliteal vein was 40%; and the sensitivity in the femoral vein was 50%. After 2 years of using this screening test with one technician and one radiologist, an improvement with this noninvasive technique was shown. However, it was found that Doppler imaging is not as sensitive as venography for detecting calf thrombi. Any imaging technique should be validated by each institution to determine the validity of the instrument and the learning curve of the technician administering the examination.     

Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.     

Prevalence of teaching apical patency and various instrumentation and obturation techniques in United States dental schools

Maintaining an open apex beyond the apical constriction with an endodontic file during canal instrumentation is a concept that has been advocated by several authors and clinicians. To ascertain the prevalence of teaching the patency concept as well as various instrumentation and obturation techniques in the United States dental schools, a survey was conducted. Forty-eight out of a total of 53 dental schools (91%) responded to the survey. Results indicate that 50% of the schools surveyed teach the concept of patency to their undergraduates or graduates or both; 83% teach a step-back instrumentation technique; and 89.6% teach lateral condensation of gutta percha as their primary obturation technique.