High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Purification, crystallisation and preliminary X-ray diffraction study of ribosome inactivating protein: saporin

OBJECTIVES: To compare transesophageal atrial pacing stress echocardiography with dobutamine stress echocardiography for feasibility, safety, duration, patient acceptance and concordance in inducing wall motion abnormalities. BACKGROUND: Transesophageal atrial pacing is an effective method of increasing heart rate and has been used in the assessment of coronary artery disease. METHODS: Both tests were performed in sequence on the same patients in random order. Transesophageal atrial pacing stress echocardiography began at a heart rate of 10 beats/min above the baseline value and was increased by 20 beats/min every two min until 85% of the age-predicted maximum heart rate or another end point was reached. Dobutamine echocardiography was performed using three-min stages and a maximum dose of 40 microg/kg per min. Atropine (total dose < or =2 mg) was administered at the start of the 40 microg/kg per min stage if needed to augment heart rate or during pacing if Wenckebach heart block occurred. RESULTS: Transesophageal atrial pacing stress echocardiography was feasible in 100 of 104 patients (96%); the duration (8.6+/-3.6 min) was significantly shorter than that of dobutamine stress echocardiography (15.1+/-3.9 min) (p = 0.0001). With transesophageal atrial pacing stress echocardiography, the recovery period was shorter, symptoms and dysrhythmias were fewer, hypertension and hypotension were less common and target heart rate was more frequently achieved. No complications occurred with either test. Patient acceptance was satisfactory. Agreement between results of both tests was good for segmental wall motion scoring with a 16-segment model, scores 1 to 5 (kappa: rest, 0.79; peak, 0.57) and test interpretation (normal, ischemia, infarction or resting wall motion abnormality with ischemia) (kappa: 0.77). CONCLUSIONS: Transesophageal atrial pacing stress echocardiography is a feasible, well-tolerated alternative to dobutamine stress echocardiography. It can be performed rapidly and shows good agreement with dobutamine stress echocardiography in the induction of myocardial ischemia.     

RS-102221: a novel high affinity and selective, 5-HT2C receptor antagonist

The 5-HT2C receptor is one of three closely related receptor subtypes in the 5-HT2 receptor family. 5-HT2A and 5-HT2B selective antagonists have been described. However, no 5-HT2C selective antagonists have yet been disclosed. As part of an effort to further explore the function of 5-HT2C receptors, we have developed a selective 5-HT2C receptor antagonist, RS-102221 (a benzenesulfonamide of 8-[5-(5-amino-2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione). This compound exhibited nanomolar affinity for human (pKi = 8.4) and rat (pKi = 8.5) 5-HT2C receptors. The compound also demonstrated nearly 100-fold selectivity for the 5-HT2C receptor as compared to the 5-HT2A and 5-HT2B receptors. RS-102221 acted as an antagonist in a cell-based microphysiometry functional assay (pA2 = 8.1) and had no detectable intrinsic efficacy. Consistent with its action as a 5-HT2C receptor antagonist, daily dosing with RS-102221 (2 mg/kg intraperitoneal) increased food-intake and weight-gain in rats. Surprisingly, RS-102221 failed to reverse the hypolocomotion induced by the 5-HT2 receptor agonist 1-(3-chlorophenyl)piperazine (m-CPP). It is concluded that RS-102221 is the first selective, high affinity 5-HT2C receptor antagonist to be described.     

Two-incision radial keratotomy for low myopia with astigmatism

PURPOSE: To evaluate the effectiveness of two-incision radial keratotomy (RK) in correcting low-magnitude refractive myopic astigmatism. SETTING: Two clinical study sites, one in St. Louis, Missouri, USA, the other in Caracas, Venezuela. METHODS: Fifty-seven eyes of 43 patients with low-magnitude myopic astigmatism had two-incision RK at one of two clinical study sites. In the initial phase of this series, 10 eyes with amblyopia at the 20/30 level had surgery at one center. Refractive keratotomy was performed with the radial incision placed in the plus cylinder axis of refraction. This axis was verified as the meridian of greatest corneal curvature by standard keratometry and computer-assisted corneal topographic analysis. Two eyes received a second operation (enhancement). RESULTS: Mean follow-up was 11.1 months (range 6 to 12 months). Mean preoperative and postoperative myopic spherical equivalent measured -1.42 diopters (D) +/- 0.51 (SD) and -0.14 +/- 0.39 D, respectively; the mean reduction was 1.28 +/- 0.59 D (P = .0001). Mean preoperative and postoperative refractive astigmatism was 1.41 +/- 0.45 D and 0.48 +/- 0.33 D, respectively (P = .0001). Mean preoperative and postoperative keratometric astigmatism was 1.26 +/- 0.54 D and 0.31 +/- 0.35 D, respectively, a mean reduction of 0.95 D (P = .0001). The surgical meridian was flattened by an average of 2.06 D by keratometry and the orthogonal meridian, by an average of 1.10 D. Preoperative uncorrected visual acuity (UCVA) was 20/40 or better in five (9%) eyes (range counting fingers to 20/40). Postoperative UCVA acuity was 20/40 or better in all eyes (mean acuity 20/25). In the nonamblyopic subgroup mean postoperative UCVA was 20/24. CONCLUSIONS: A limited number of radial incisions placed in the topographically confirmed axis of greatest curvature are effective in the treatment of low-magnitude myopic astigmatism.     

Overview and implications of Medicaid managed care for people with developmental disabilities

The inclusion of people with developmental disabilities in managed care as part of general efforts by states to enroll and Medicaid recipients in such plans was reviewed. Managed care was defined and the processes by which managed care organizations deliver services were explained. Escalating costs and utilization were discussed as the primary reason for the shift to managed care. The use of Medicaid Section 1115 waivers by states to include Medicaid recipients was explored. The relation between acute health care and long-term care, and the utilization patterns in each, were briefly described. Finally, elements of managed care that are particularly important to people with developmental disabilities, such as care coordination, maintenance of quality, and individual and family support, were discussed.     

Bone tumors of the coracoid process of the scapula

Bone tumors of the coracoid process of the scapula are rare, and diagnosis and treatment often are delayed. The records of 18 patients with bone tumors of the coracoid process were reviewed. Histologic types included eight cases of ordinary chondrosarcoma, three cases of dedifferentiated chondrosarcoma, two cases of osteoid osteoma, and one case each of osteosarcoma, plasmacytoma, lymphoma, giant cell tumor, and aneurysmal bone cyst. All 18 patients had shoulder pain, and eight of them had been treated with steroid injections for nonneoplastic conditions. Radiologically, chondrosarcoma did not always show clear cortical destruction, and one giant cell tumor had features mimicking those of chondrosarcoma. Five patients (three with dedifferentiated chondrosarcoma, one with chondrosarcoma, one with plasmacytoma) died of disease. The coracoid process was the site with a markedly high proportion of chondrosarcomas. Bone tumors of the coracoid process may be difficult to detect on plain radiographs. In the patient with persistent shoulder pain unresponsive to the selected treatment, additional imaging studies should be considered to eliminate the possibility of a bone lesion.     

Genomic variation of human papillomavirus type 16 and risk for high grade cervical intraepithelial neoplasia

BACKGROUND: Epidemiologic studies have demonstrated strong and consistent associations between the detection of human papillomavirus (HPV) type 16 DNA and the risk of cervical intraepithelial neoplasia (CIN) and cervical cancer. However, HPV16 is also the most common type of HPV in the normal population, and only a minority of women with HPV16 infection develop cervical cancer. Studies of genomic heterogeneity in HPV16 have demonstrated the presence of multiple variant forms in all human populations examined to date. It is conceivable that the natural variants of HPV16 in a given population may not have the same biologic behavior. PURPOSE: This study was designed to determine the association between natural variants of HPV16 and the risk of biopsy-confirmed CIN 2 or 3, the most important precancerous lesions of the uterine cervix. METHODS: Prospective studies were conducted among 1) women attending a university and 2) women presenting to a sexually transmitted disease clinic. Subjects were eligible for inclusion in this investigation if the initial cytologic findings did not reveal CIN 2-3 and HPV16 DNA was detected by means of a polymerase chain reaction (PCR)-based method in one or more cervical or vulvovaginal samples. Eligible subjects were followed every 4 months with cervical Pap smears and colposcopic examinations. Women were referred for biopsy if cytology or colposcopy suggested CIN 2-3. Two groups of HPV16 variants, prototype-like and nonprototype-like, were determined by means of single-strand conformation polymorphism (SSCP) analysis of PCR products from the noncoding region of the viral genome. Representative SSCP patterns from HPV16 variants were further characterized by direct DNA sequencing of the PCR products. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated by Cox regression analysis. RESULTS: Prototype-like variants accounted for 79% of the HPV16 detected in university students and 86% of the virus detected in patients presenting to the sexually transmitted disease clinic. CIN 2-3 was confirmed by biopsy in nine of 57 HPV16-positive women attending the university and in 10 of 66 HPV16-positive women presenting to the sexually transmitted disease clinic. Among university students, those with HPV16 nonprototype-like variants were 6.5 (95% CI = 1.6-27.2) times more likely to develop CIN 2-3 than those with prototype-like variants. A similar association was observed among women presenting to the sexually transmitted disease clinic (RR = 4.5; 95% CI = 0.9-23.8). CONCLUSIONS: This study suggests that the risk of developing CIN 2-3 is not the same with all variants of HPV16 and that nonprototype-like variants confer a greater risk compared with prototype-like variants. The important genomic differences underlying this increased risk of CIN 2-3 remain to be determined.