C-terminal hybrid mutant of Bacillus pumilus cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization.

AIMS: To investigate the impact of the highly variable C-terminal domain of cyanide dihydratase, a member of the nitrilase superfamily, on its activity and stability. METHODS AND RESULTS: Generating and analysing the thermal stability and pH tolerance of chimeric cyanide dihydratase proteins has provided a platform to investigate domains within the C-terminus and their effect on quaternary structure of the protein. The protein oligomerization state was inferred from native protein size by gel exclusion chromatography. CONCLUSIONS: Our data indicates that the influence of the cyanide dihydratase C-terminus on thermal stability stems from its participation in oligomerization at the major C-surface interface. The formation of this surface is crucial for the activity and stability of CynD. Gel filtration chromatography of an N-terminal deletion mutant, CynDpum ∆303, revealed a defect in oligomerization, and another mutant R67C was suppressed by introduction of a heterologous C-terminus as a chimeric protein. This indicates that the C-terminus from Pseudomonas stutzeri stabilizes CynD by supporting oligomerization between dimers at the C-surface. The chimeric protein CynDpum-stut exhibited full activity at pH 9, a pH where the parent enzyme is nearly inactive, and retained 40% of its activity at pH 9·5 making it a unique pH tolerant mutant. SIGNIFICANCE AND IMPACT OF THE STUDY: The study characterized a chimeric protein with remarkable thermal stability and tolerance to alkaline conditions, features essential for practical application as industrial cyanide solutions are maintained as highly alkaline solutions to prevent formation of hydrogen cyanide gas.

Predictors of length of stay after coronary stenting.

BACKGROUND: Postprocedure length of stay (LOS) remains an important determinant of medical costs after coronary stenting. Variables that predict LOS in this setting have not been well characterized. METHODS: We evaluated 359 consecutive patients who underwent coronary stenting with antiplatelet therapy. Sequential multiple linear regression (MLR) models were constructed with use of 4 types of variables to predict log-transformed LOS: preprocedure, intraprocedure, and postprocedure factors and adverse outcomes. RESULTS: Preprocedure factors alone explained more than one third of the variability in postprocedure LOS (adjusted R(2) = 0.37). The addition of procedural variables added little to the model (adjusted R(2) = 0.39). Entering nonoutcome postprocedure variables significantly enhanced the predictive capacity of the model, explaining more than half the variability in postprocedure LOS (adjusted R(2) = 0.54). In the final model, addition of outcome variables increased its predictive capacity only slightly (adjusted R(2) = 0.61). In this model, significant preprocedure factors included: myocardial infarction (MI) within 24 hours, MI within 1 to 30 days, women with peripheral vascular disease, intravenous heparin, and chronic atrial fibrillation. High-risk intervention was the only significant intraprocedure variable. Significant postprocedure factors included periprocedure ischemia; cerebrovascular accident or transient ischemic attack; treatment with intravenous heparin or nitroglycerin or intra-aortic balloon pump; and need for blood transfusion. Significant adverse outcomes included contrast nephropathy, gastrointestinal bleeding, arrhythmia, vascular complication, and repeat angiography. CONCLUSION: This prediction model identifies a number of potentially reversible factors responsible for prolonging LOS and may enable the development of more accurate risk-adjusted methods with which to improve or compare care.

Processing demands during auditory learning under degraded listening conditions.

This investigation employed measures of learning accuracy (performance) and learning ease (attention or psychological effort) to assess processing demands during auditory learning under degraded listening conditions. Learning accuracy was measured with a highly intelligible paired-associate learning task presented to 49 normal-hearing adults under different signal-to-competition ratios and signal presentation levels. Learning ease was assessed by a simultaneously presented probe reaction-time task. Final results indicated that (1) primary signal presentation level exerted no effect either on learning accuracy or ease, and (2) the introduction of competing speech into the listening environment exerted no effect on learning performance, but resulted in a significant increase in learning effort. These findings have important implications for listening conditions in educational settings, hearing aid selection, education of hearing-impaired and learning-disabled children, and future study of attentional demands during auditory processing.

The hyperactive acoustic reflex. Four case studies.

Four case studies are presented to illustrate that low acoustic reflex thresholds may have value in assessment of central auditory processing disorders. Neurologic, electronystagmographic, EEG, speech pathologic, and/or psychological evaluations suggested CNS impairment. Tests for central auditory processing disturbances were positive. For three subjects, acoustic reflex responses were elicited at unusually low hearing threshold levels. A fourth subject showed lowering of acoustic reflex thresholds following bilateral electroshock treatment. These lowered thresholds were interpreted as hyperactive acoustic reflex responses representing a decreased central inhibition on peripheral auditory function. It is proposed that presence of hyperactive acoustic reflex responses may be associated with a concomitant central auditory processing disorder.

Detection of Chlamydia trachomatis endocervical infections by ligase chain reaction versus ACCESS Chlamydia antigen assay.

Ligase chain reaction (LCR) was compared with ACCESS immunoassay for detection of chlamydial infections in females. Despite efforts to improve ACCESS performance by evaluation of specimens that were in the test performance "grey zone," LCR remained more sensitive and was less expensive to perform. ACCESS had a sensitivity of 83.9%, a specificity of 99.7%, a positive predictive value of 96.3%, and a negative predictive value of 98.5%.