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.     

Low-frequency suppression of auditory nerve responses to characteristic frequency tones

The effects of low-frequency (50, 100, 200 and 400 Hz) 'suppressor' tones on responses to moderate-level characteristic frequency (CF) tones were measured in chinchilla auditory nerve fibers. Two-tone interactions were evident at suppressor intensities of 70-100 dB SPL. In this range, the average response rate decreased as a function of increasing suppressor level and the instantaneous response rate was modulated periodically. At suppression threshold, the phase of suppression typically coincided with basilar membrane displacement toward scala tympani, regardless of CF. At higher suppressor levels, two suppression maxima coexisted, synchronous with peak basilar membrane displacement toward scala tympani and scala vestibuli. Modulation and rate-suppression thresholds did not vary as a function of spontaneous activity and were only minimally correlated with fiber sensitivity. Except for fibers with CF < 1 kHz, modulation and rate-suppression thresholds were lower than rate and phase-locking thresholds for the suppressor tones presented alone. In the case of high-CF fibers with low spontaneous activity, excitation thresholds could exceed suppression thresholds by more than 30 dB. The strength of modulation decreased systematically with increasing suppressor frequency. For a given suppressor frequency, modulation was strongest in high-CF fibers and weakest in low-CF fibers. The present findings strongly support the notion that low-frequency suppression in auditory nerve fibers largely reflects an underlying basilar membrane phenomenon closely related to compressive non-linearity.     

Sj?gren''s syndrome in patients with newly diagnosed untreated non-Hodgkin''s lymphoma

A prfA gene encoding polypeptide release factor RF1 was cloned from Thermus thermophilus. T thermophilus RF1 shares 68% homology with Escherichia coli RF1, and its overproduction reduced readthrough translation of UAG, not of UGA, in the lacZ gene. Rapid purification of T thermophilus RF1 was achieved by T7-RNA polymerase driven overexpression of T thermophilus RF1 protein with a C-terminal histidine tag.     

Superconducting gap function and upper critical field in the presence of inhomogeneous magnetic order

Rare earth ternary superconductors are known to exhibit oscillatory magnetic orders below their superconducting transition temperatures. The study of behaviour of superconducting electrons in an inhomogeneous magnetic field is therefore important for such systems. We report here the results of our theoretical study of superconducting gap function Δ(T) and upper critical fieldH _c2 (T). The results are applied to analyse and explain the variation of Δ(T) andH _c2 (T) in case of NdRh₄B₄.     

Update on Alzheimer''s disease

The nation's health care system is undergoing a period of rapid change that will profoundly affect women's health care services and, ultimately, women's health. Although managed care is quickly becoming the predominant mode of health care delivery in the United States, a new, more consumer-focused, and accountable model known as organized systems of care (OSC) is emerging. OSC development has been driven by large private and public employers seeking to purchase the highest quality health care for the best price. The changes in health care delivery encouraged by these innovative employers will provide women with optimal care and attention, which will in turn help them attract and retain a competitive and productive workforce.     

Continuous measurements of cardiac output in the perioperative period

Management of critically ill patients is based on knowledge of fundamental physiological variables. Automatized and continuous measurement of these variables is preferable. A new system based upon the thermodilution method has been developed to measure cardiac output automatically and continuously. We evaluated the system in the potentially unstable perioperative period with possible great and rapid changes in cardiac output. Twenty patients, scheduled for open heart or abdominal aortic aneurysm surgery, were included into the study, which was approved by the local ethical committee. The patients were monitored for up to 30 hours. At random intervals five iced bolus thermodilution cardiac output (BCO) determinations were made and compared to the continuous measurements (CCO). Two hundred and thirty-one pairs of data were obtained. The cardiac outputs ranged from 2.5-14.9 l/min. The absolute bias was 0.31 l/min (95% limits of agreement -1.4 l/min to 2.0 l/min). The mean relative error was 4.7% with a standard deviation of the relative error of 15.4%. The linear regression was represented by: CCO = 11.352 x BCO - 0.36. The correlation coefficient R was 0.90 (p < 0.001). In conclusion, the CCO measurement technique is a promising clinical method. The method is straightforward, requires no calibration, is independent of vascular geometry and measures with its limitations volumetric flow. Finally automatic and continuous patient monitoring provides more information and has potential to reveal previously undetected haemodynamic events.     

Vasopeptidase inhibitors: incorporation of geminal and spirocyclic substituted azepinones in mercaptoacyl dipeptides

A series of 7-(di)alkyl and spirocyclic substituted azepinones were generated and incorporated as conformationally restricted dipeptide surrogates in mercaptoacyl dipeptides. Clear structure-activity relationships with respect to both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) activity in vitro were observed. The best in this series, compound 1g, a geminally dimethylated C-7-substituted azepinone, demonstrated excellent blood pressure lowering in animal models. Compound 1g (BMS-189921) is characterized by a good duration of activity and excellent oral efficacy in models relevant to ACE or NEP inhibition, and its activity is comparable to that of the clinically efficacious agent omapatrilat. Consequently this inhibitor has been advanced clinically for the treatment of hypertension and congestive heart failure.     

Using fluorescence-based human androgen receptor gene assay to analyze the clonality of microdissected dendritic cell tumors

The AGT1 permease is a alpha-glucoside-H+ symporter responsible for the active transport of maltose, trehalose, maltotriose, alpha-methylglucoside, melezitose and sucrose. In wild-type as well as in MAL constitutive strains, alpha-methylglucoside seemed to be the best inducer of transport activity, while trehalose had no inducing effect. Based on the initial rates of transport it seems that the sugar preferentially transported by this permease is trehalose, followed by sucrose.