The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-in-activating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A1 form of Slt-IA (wild-type-delta). The same general relationships held: relative to wild type-delta, Y114F-delta was attenuated about 7-fold, and Y114S-delta about 300-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Setting the record straight.

Replies to comments by H. W. Marsh and L. A. Roche (see record 1997-43129-003) on the author's article (Educational Policy Analysis Archives, 1997) on student evaluation and academic freedom. The author expresses his dismay that Marsh and Roche misrepresented (not misinterpreted) the article without documenting their misrepresentation. A documented response to this undocumented misrepresentation is provided. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.     

Recent thinking about the concept of alcohol dependence

This paper reviews past definitions of alcohol dependence as well as that presently adopted in the 4th edition of the Diagnostic and Statistical Manual of the American Psychiatric Association. The role of alcohol consumption in diagnostic criteria and the measurement of various dimensions of drinking (frequency, usual quantity per occasion, overall volume) are also discussed. Reliable criteria for identifying both alcohol abuse and dependence are available. Methodology for assessment of different dimensions of drinking behavior also exists. Epidemiologists examining the role of alcohol in health problems should consider the diagnostic category and the dimensions of drinking behavior that are important to their study. Measurements should be adopted in accordance with this assessment.     

Verification of ANSI C37.90.1 SWC test generator outputs

Awareness of fast transient events in the electrical power environment has increased with the release of related test standards (ANSI C37.90.1 and IEC 801-4) and with increased usage of solid state circuits in utility equipment. Reliable measurement of the fast transient test waveforms and repeatable performance of test setups are persistent problems for equipment manufacturers and users. The construction, characterization, and usage of two high voltage test dividers for measurement of fast transient test waveforms are described. Measurements on a C37.90.1 (SWC) test generator are presented along with tutorial background. Proposals for changes to the ANSI C37.90.1 specification are included     

Comparison of smoothness evaluation devices for power cablecompounds

A variety of data from different devices has been generated on the smoothness of shield products, but no direct comparison of compound smoothness for the devices themselves has been made. The purpose of this study was to compare four different methods for evaluating the smoothness of power cable compounds. The methods compared include: image analysis, laser scanning, mechanical profilometry and photomicroscopy with visual rating. Extruded tape samples of conductor shields, filled insulations and strippable shields were evaluated     

A bidding decision index for construction contractors

Results from applying the model on a sample of contractors, the majority of whom were international and operating in Egypt, reinforces the credibility of the developed methodology, claim the authors.