Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.     

Sulfated zirconia catalysts: Are Brønsted acid sites the source of the activity?

The thermal decomposition products of pyridinium sulfate differ from those of pyridinium sulfate supported on zirconia which in turn differs from that of pyridine adsorbed on a sulfated zirconia. Unsupported pyridinium sulfate decomposes to produce pyridine and sulfuric acid, and these subsequently react to produce oxides of carbon and sulfur. Zirconia that is sulfated and then exposed to pyridine does not release detectable amount of pyridine during heating in an inert gas; rather the pyridine undergoes oxidation reduction reactions simultaneously to release CO₂ and sulfur compounds. Pyridinium sulfate supported on zirconia decomposes upon heating to release pyridine and sulfuric acid, which reacts with the zirconia. The desorption of pyridine in one case and only CO₂/SO_x in the other case suggests that sulfated zirconia does not contain Brønsted acidity that can form pyridinium sulfate.     

Current practice in software quality and the impact of certification schemes

The paper presents the findings of a survey that investigated the level of quality management practice within some 150 UK companies from a sample of 500. It provides a snapshot of practice at the time of the survey, and assesses the impact of government quality initiatives particularly, the TickIT scheme at that time. The survey methodology is described, together with the results and conclusions. The sample has been graded by size of company, which the authors consider to have a significant effect upon the adoption of quality practices. The survey highlights the need to encourage small companies to adopt quality practices and to assist them with the short-term costs incurred.     

Efficient random subcloning of DNA sheared in a recirculating point-sink flow system

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.     

The effect of topical corticosteroids on refractive outcome and corneal haze after photorefractive keratectomy

BACKGROUND: The effect of topical corticosteroids after excimer laser photorefractive keratectomy (PRK) remains a matter of some controversy. Refractive effects may be different according to the amount of myopia and timing of instillation. METHODS: Two groups of patients were studied: Study A consisted of 215 eyes (128 patients) with PRK (mean baseline myopia, -6.53 +/- 2.22 D) that received no corticosteroids (No Corticosteroid Group) unless significant regression or corneal haze appeared (Delayed Corticosteroid Group), and in Study B, we randomly assigned eyes to the Initial Corticosteroid Group (mean baseline myopia, -6.39 +/- 1.84 D) or the No/delayed Corticosteroid Group (mean baseline myopia -5.78 +/- 2.02 D). Clinical results after PRK for low-to-moderate and high myopia were compared. RESULTS: In the first group, 70.9% (73 eyes) of moderately myopic eyes (mean, -4.56 +/- 1.10 D) belonged to the No Corticosteroid Group that had a mean refraction of -5.39 +/- 1.77 D. Delayed Corticosteroid Group eyes were more myopic (mean, -7.52 +/- 2.10 D), and showed more severe haze than those in the No Corticosteroid Group. In study B, only in high myopes with more than -6.00 D (mean, -7.76 +/- 1.15 D) did refraction and corneal haze outcomes show significant difference between the Initial Corticosteroid Group and the No/delayed Corticosteroid Group. CONCLUSIONS: The effects of topical corticosteroids after PRK were less in moderate myopes compared to high myopes. Delayed instillation of corticosteroids did not reverse the regression or haze whereas initial instillation showed a beneficial effect on high myopes but not on moderate myopes.     

Comments on “Editorial: fuzzy models-what are they andwhy?”

In the editorial by J.C. Bezdek (ibid., p.1), an example is presented to demonstrate differences between fuzzy membership and probability. The authors argue that probability can be used in a way much more closely analogous to this use of fuzzy membership, weakening the argument for the latter     

The use of stokeslets to describe the arbitrary translation of a disk near a plane wall

A previously presented method is extended to describe the fully three-dimensional Stokes flow generated by the translation in any direction of an arbitrarily oriented disk in fluid bounded by a plane wall. The velocity field is represented solely in terms of stokeslet distributions on the disk, modified to take account of the bounding wall according to the century-old idea of Lorentz. Sets of integral equations of the second kind, not all disjoint, are obtained for the Abel transforms in each Fourier mode of the density functions. However, only a few modes need be considered in determining the flow field to order D ^-3, where D is the distance of the disk axis from the wall. Less detail is required to evaluate the drag force and torque experienced by the disk.     

A multi-agent decision support system for stock trading

A distributed problem solving system can be characterized as a group of individual cooperating agents running to solve common problems. As dynamic application domains continue to grow in scale and complexity, it becomes more difficult to control the purposeful behavior of agents, especially when unexpected events may occur. This article presents an information and knowledge exchange framework to support distributed problem solving. From the application viewpoint the article concentrates on the stock trading domain; however, many presented solutions can be extended to other dynamic domains. It addresses two important issues: how individual agents should be interconnected so that their resources are efficiently used and their goals accomplished effectively; and how information and knowledge transfer should take place among the agents to allow them to respond successfully to user requests and unexpected external situations. The article introduces an architecture, the MASST system architecture, which supports dynamic information and knowledge exchange among the cooperating agents. The architecture uses a dynamic blackboard as an interagent communication paradigm to facilitate factual data, business rule, and command exchange between cooperating MASST agents. The critical components of the MASST architecture have been implemented and tested in the stock trading domain, and have proven to be a viable solution for distributed problem solving based on cooperating agents