Eukaryotic arylamine N-acetyltransferase. Investigation of substrate specificity by high-throughput screening

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.     

Sustained complete remission of metastatic dermatofibrosarcoma protuberans with imatinib mesylate

Dermatofibrosarcoma protuberans (DFSP) is a soft tissue tumor which may recur locally and rarely causes metastases to vital organs. DFSPs have specific chromosomal abnormalities involving the platelet-derived growth factor beta-chain locus (PDGFB) which may render these tumors responsive to targeted therapy with the tyrosine kinase inhibitor imatinib mesylate. A patient with locally recurrent and metastatic DFSP resistant to first-line chemotherapy was treated with imatinib mesylate 400 mg/day. The tumor was examined by a novel fluorescence in situ hybridization (FISH) method for specific rearrangements of the PDGFB locus. The patient was followed for response and toxicity by physical examination and imaging studies. FISH revealed PDGFB rearrangement indicative of multiplication of the PDGFB fusion locus within a ring chromosome. Physical examination showed response within the first month of treatment, and subsequent computed tomography and fluorodeoxyglycose positron emission tomography documented complete response to imatinib therapy. Our patient is now in sustained complete remission for 20 months with minimal toxicity. We conclude that sustained complete remission of metastatic DFSP with specific FISH abnormalities involving the PDGFB locus can be obtained with imatinib mesylate with minimal toxicity for the patient.     

Pars plana vitrectomy in patients with intermediate uveitis

Objective: To describe the effect of pars plana vitrectomy in patients with intermediate uveitis. Methods: Retrospective analysis of the clinical course and visual outcome following pars plana vitrectomy in patients with intermediate uveitis. Results: Thirty-two patients (43 eyes) were included in the study. Pars plana vitrectomy was combined with cataract surgery in 22 of 43 eyes. The intermediate uveitis was associated with sarcoidosis in 16 eyes and multiple sclerosis in five eyes, and was idiopathic in 22 eyes. The mean (±SD) follow-up was 45.6 (±38) months (range: 6–146 months). In 19 of 43 eyes (44.1%), there was improvement in the course of uveitis, allowing the discontinuation of immunosuppressive treatment in seven patients. Cystoid macular edema resolved in 12 of 37 eyes (32.4%). Forty of 43 eyes achieved a better or retained their initial visual acuity. The remaining three eyes deteriorated by two or more lines in the Snellen chart due to the progression of cataract, chronic cystoid macular edema, and glaucomatous optic atrophy, respectively. Conclusions: The results of this study suggest that pars plana vitrectomy may have a beneficial effect on the course of uveitis and the associated complications of cystoid macular edema, thereby reducing the need for long-term immunosuppression. Pars plana vitrectomy combined with simultaneous cataract surgery can improve the visual outcome in these patients.     

Long-term immunosuppressive treatment of serpiginous choroiditis

Objective: To determine the efficacy of immunosuppressive treatment in serpiginous choroiditis. Design: The clinical courses were reviewed of six consecutive patients (12 eyes) with vision-threatening, steroid-dependent/resistant serpiginous choroiditis treated with a combination of immunosuppressive agents including azathioprine, cyclosporine, and cyclophosphamide. All patients underwent treatment for at least 12 months. Results: The follow-up period ranged from 17 to 105 months (mean 57, median 43). All patients were able to taper oral steroids. Five patients discontinued all immunosuppressive medications after a treatment period of 12 to 69 months (mean 39 months). Immunosuppressive treatment was continued in one patient at a ‘low’ maintenance dose. Ten eyes had improved visual acuities, while vision remained impaired in two due to macular scars. Recurrence was noted in two patients when an attempt was made to decrease the dose of immunosuppressive medication. Two patients experienced side effects which were reversed by decreasing the dose of the medications. Conclusion: Long-term immunosuppressive treatment appears to prolong remission and preserve vision in patients with serpiginous choroiditis.     

Outcomes of Treatment with Immunomodulatory Therapy in Patients with Corticosteroid-Resistant Juvenile Idiopathic Arthritis-Associated Chronic Iridocyclitis

Purpose: To describe the clinical outcome of patients with juvenile idiopathic arthritis (JIA)-associated chronic iridocyclitis unresponsive to conventional therapy, in whom inflammation was eventually controlled using immunomodulatory therapy (IMT), and to determine if patients treated early with IMT have better visual acuity outcomes than those treated with corticosteroid alone. Methods: Patients with JIA-associated chronic iridocyclitis receiving immunomodulatory therapy at the Ocular Immunology and Uveitis Service of the Massachusetts Eye and Ear Infirmary between 1981 and 2001 were studied. Inclusion criteria: JIA with chronic corticosteroid-dependent uveitis; IMT for a minimum of one year; minimum follow-up of three years; inflammation eventually controlled for a minimum of six consecutive months by IMT. Results: At last visit, 51% of eyes (23 eyes) had acuity of 20/20 to 20/40, 16% (7 eyes) had 20/50 to 20/100, and 33% (15 eyes) had 20/200 or less. In patients whose chronic inflammation was eventually controlled within three years from onset of the uveitis, final bilateral vision was maintained within 20/20 to 20/30, except for a patient whose one eye had poor vision at initial consultation. When acuities at the endof the follow-up period of patients treated early with IMT were compared with acuities at the initial consultation of patients treated late with IMT, with duration of uveitis matched for each patient, visual acuities of those treated early were statistically significantly better than those treated late with IMT (p < 0.005 right and left eyes pooled; p = 0.0075 best eyes; p = 0.0375 worst eyes). Conclusions: We noted improvement or maintenance of visual acuity (86%) during the course of follow-up of patients with treatment-resistant JIA-associated uveitis treated with effective IMT. However, only IMT given early in the disease course was noted to be associated with bilateral visual acuity of 20/30 or better.     

Biliary stenting alone versus biliary stenting plus sphincterotomy for the treatment of post-laparoscopic cholecystectomy biliary leaks: a prospective randomized study

OBJECTIVE: Although various endoscopic techniques have been proved effective in treating post-cholecystectomy biliary leaks, the choice of the best method remains controversial. The aim of this prospective study was to compare the efficacy and safety of biliary stenting alone with biliary stenting plus sphincterotomy for the treatment of post-cholecystectomy biliary leaks. METHODS: Patients with post-laparoscopic cholecystectomy leaks were randomized into two groups. The first group included 24 patients who were treated with a 7 Fr biliary stent alone, and the second group included 28 patients who underwent an endoscopic sphincterotomy followed by insertion of a 10 Fr biliary stent. RESULTS: Endoscopic therapy was successful in all patients (100%). Clinical improvement was observed after 2-6 days. Patients remained hospitalized for 4-12 days. Stents were removed after 6.7 (6-8) weeks. The overall complication rate was 4.16% for the first group and 10.71% for the second (P=0.615). No complications were recorded during the follow-up period. CONCLUSIONS: Endoscopic therapy of biliary leaks with a small-diameter biliary stent alone is as effective and safe as endoscopic sphincterotomy followed by insertion of a large-diameter stent.