Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.     

Contactless method of determining the coefficient of thermal diffusivity of local domains of surface layers and thin films

A method is proposed for measuring the coefficient of thermal diffusivity of microsections of surface layers and thin films. The coefficient of thermal diffusivity is calculated from the time dependence of the heat flux emitted by the heated surface.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 31, No. 2, pp. 289–294, August, 1976.     

Electrical and structural properties of epitaxial CdTe/HgCdTe interfaces

In this study, CdTe epilayers were grown by metalorganic chemical vapor deposition on epitaxial HgCdTe with the purpose of developing suitable passivation for HgCdTe photodiodes. Two types of CdTe layers were investigated. One was grown directly,in situ, immediately following the growth of HgCdTe. The second type of CdTe was grown indirectly, on top of previously grown epitaxial HgCdTe samples. In this case, the surface of the HgCdTe was exposed to ambient atmosphere, and a surface cleaning procedure was applied. The material and structural properties of the CdTe/HgCdTe interfaces were investigated using secondary ion mass spectroscopy, Auger electron spectroscopy, Rutherford back scattering, and x-ray double crystal diffractometry techniques. Electrical properties of the CdTe/HgCdTe heterostructure were determined by capacitance-voltage (C-V) characterization of Schottky barrier devices and metal insulator semiconductor devices. Also, a preliminary current-voltage characterization of n⁺ p photodiodes was performed. A theoretical model suitable for analysis of graded heterojunction devices was used for interpretation of C-V measurements.     

Free-standing optical filters for a nanolithography source operating in the 12–15 nm wavelength range

Optical filters in the form of free-standing multilayer films have been obtained for a nanolithography source operating in the 12–15 nm range of wavelengths. A method for the synthesis of such filters is described.     

An improved method for the determination of adenosinetriphosphate in environmental samples

An improved method has been developed for the analysis of the adenosinetriphosphate (ATP) content of environmental samples with the luciferin-luciferase assay. Factors affecting the recovery and stability of the ATP as well as the enzymatic reaction were optimized. The most versitile method utilizes procedure which can be used for natural waters, sediments and sludges. This involves homogenization at room temperature in 10⁻² M trisodium phosphate, pH 11.7, containing 23% chloroform. This method is more effective than other methods tested and results in minimal interference with the enzymatic reaction. It also provides improved ATP recovery and long term stability of extracted ATP. Another method that can be used on natural waters and sludges is by boiling for 5 min in 10⁻² M glycine buffer, 5 × 10⁻³ M Mg-EDTA, pH 10, which resulted in complete extraction and no hydrolytic loss of ATP. However, it is not satisfactory for sediments containing high humic acid substances as well as high metal loadings.In addition to the direct comparison of endogenous ATP extraction by various methods, actual recoveries of spiked radioactive ATP was measured. ATP adsorption, hydrolysis and recoveries in each method were determined by the use of [8-¹⁴C] ATP and [γ-³²P] ATP.     

Platelet monoamine oxidase (MAO) activity: Personality, substance use, and the stress-response-dampening effect of alcohol.

The platelet MAO activity of 557 male Ss (aged 21–26 yrs) was assessed with substance use and abuse, family history of alcoholism, and personality traits. Low platelet MAO activity was associated with (1) undercontrolled behavioral style as evidenced by high scores on measures of antisociality, (2) increased use of cigarettes, and (3) increased rates of illicit drug use and adverse consequences of drug use. Low MAO activity was not related to either alcohol use or abuse. In an experimental study, 17 Ss with MAO activity in the lowest decile and 38 Ss with MAO activity in the highest 2 deciles were administered either a .625 g/kg dose of alcohol or a placebo and exposed to an anxiety-provoking stressor. Low-MAO-activity Ss appeared to show stronger stress response dampening on heart rate than high-MAO-activity Ss. No other differences in response to alcohol were found, and MAO activity was not related to general reactivity to the stressor. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Obituaries: Lizette Peterson (1951-2002).

Reports the death of Lizette Peterson (1951-2001). Her various contributions to the fields of pediatric and clinical psychology are noted. (PsycINFO Database Record (c) 2010 APA, all rights reserved)