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.     

消泡剂、增稠剂和有机硅添加剂

1消泡剂对涂料来说，起泡总是要不得的。起泡的问题在涂料制造过程本身中就是存在的。起泡使投料量不得不降低，设备有效利用率不得不减少。但起泡的问题更多地发生在施工过程中，使漆膜出现种种弊病。而且，起泡造成的不仅仅限于对视觉的干扰，还会使涂层的保护功能难以正常发挥。于是，在涂料配方中，消泡剂就是一个不可缺少的组成成分几乎所有的涂料体系的组成成分都会对起泡现象发生影响，有的影响是正面的，有的是反面的。此外，基材性质和施工方法对起泡的表现也是有影响的。必须认识到，消泡在很大程度上是一个有着极强针对性的问题…     

Diabetes and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS): radiolabeled polymerase chain reaction is necessary for accurate detection of low percentages of mutation

A 6-yr-old boy presented with muscle weakness, lactic acidemia, and insulin-dependent diabetes mellitus (IDDM). Using PCR and restriction enzyme analysis, he was found to have the classical A3248G mitochondrial DNA (mtDNA) mutation frequently associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). The mutation was confirmed by sequencing muscle mtDNA. The mutation in mtDNA from muscle, lymphoblasts, and blood was clearly demonstrable by standard methods using ethidium bromide staining. His mother also had IDDM, but no A3243G mutation could be detected in her blood or transformed lymphoblasts using the same PCR technique. When PCR was carried out in the presence of [32P]deoxycytidine triphosphate, subsequent autoradiography detected the presence of the mutation at low levels in mtDNA from the mother's lymphoblasts and blood. Study of the mother's muscle showed a mitochondrial myopathy, despite the fact that she was asymptomatic. We emphasize that the increased sensitivity of radiolabeled PCR may be necessary to detect small percentages of heteroplasmic A3243G mtDNA mutation in blood from diabetic subjects. Otherwise the incidence of mtDNA mutations in both IDDM and non-insulin dependent diabetes may be underestimated.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: application of the 1H 3D-NOE/2QC experiment

A mixed general linear model analysis of the development of sleep-wake states was conducted on 37 high-risk preterm infants and replicated with a second cohort of 34 infants. Most dependent variables showed significant development over the preterm period: active sleep decreased, and active waking, quiet waking, and the organization of active sleep and quiet sleep increased over the preterm period in both cohorts. The amount of quiet sleep also increased over age, but this change was significant only for Cohort 1. Seven infant characteristics used as covariates had only minor effects. There were no significant differences in the developmental trajectories (slopes) of the two cohorts. The amounts of four variables differed between cohorts: Cohort 2 infants had less sleep-wake transition, more active sleep, less active sleep without REM, and more regular quiet sleep. These findings suggest that developmental patterns of sleep wake states are stable enough in the preterm period that deviant individual patterns might be used to identify infants with neurological problems.     

Left atrial appendage flow velocity and spontaneous echo contrast in patients with rheumatic mitral stenosis: a multiplane transesophageal echocardiographic study

BACKGROUND: Left atrial spontaneous echo contrast (LASEC), a putative marker of thrombo-embolic risk, is commonly located in the left atrial appendage (LAA). The aims of this work were to evaluate, using multiplane transesophageal echography, the echocardiographic determinants, specifically LAA outflow Doppler velocity, in the presence of SEC in patients with rheumatic MS. METHODS: Transthoracic and transesophageal echocardiographic tests were performed on 61 patients. The patients were divided into 3 groups based on the presence and type of valvular disease. Patients in group I (n = 28) presented with rheumatic mitral stenosis (MS). Patients in group II (n = 18) presented with valvular heart disease other than MS, and patients in group III (n = 15) had no history of valvular heart disease. The left atrium and appendage were examined for the presence of spontaneous echocontrast and thrombus, using multiplane echo scopy with transducer rotation. Minimal and maximal appendage areas were measured, on a computer-assisted bablet, by tracing a line from the top of the limbus of the left upper pulmonary vein to the appendage endocardial border. The LAA ejection fraction was calculated according to the formula: (maximal area-minimal area)/maximal area. Mitral valvular condition was evaluated with transthoracic and transesophageal echocardiography. Left atrial appendage blood flow velocity profiles were obtained with pulsed-wave Doppler at the orifice of the LAA. RESULTS: LASEC was present in 18 of 28 patients with mitral stenosis (64.3%). Patients with LASEC showed a greater incidence of atrial fibrillation (14/18 vs 12/43, p < 0.005), larger LAD (53.67 +/- 8.74 vs 40.54 +/- 14.85, p < 0.005), smaller LAAEF (38.7 +/- 1.53 vs 69.5 +/- 24.0, p < 0.05), smaller LAAMEV (20.28 +/- 10.07 vs 2.95 +/- 25.11, p < 0.005) and smaller LAAMFV (24.6 +/- 12.23 vs 36.00 +/- 11.01, p < 0.01), when compared with patients without LASEC. For group I, LAAEF, LAAMEV and LAAFV were smaller in patients with SEC than in patients without SEC (p < 0.005, p < 0.05, p < 0.01). However LAD values were similar for patients with and without SEC (53.67 +/- 8.75 vs 54.20 +/- 18.81, p = NS). Both LAAMEV and LAAMFV were related to SEC in patients with atrial fibrillation. However, LAD did not show the same trend. CONCLUSIONS: LASEC is more commonly observed in patients with rheumatic mitral stenosis or atrial fibrillation. Both LAAMEV and LAAMFV are associated with SEC in these patients.