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.     

Human stem cell assays in immune-deficient mice

Much of our understanding of the organization of the cells that comprise the hematopoietic system and the cellular and molecular mechanisms that regulate their development is derived from mouse models. However, knowledge of the human hematopoietic system and identification of human stem cells have, until recently, been hampered by the absence of in vivo assays that measure their repopulation capacity. The development of methods to transplant normal and leukemic human hematopoietic cells into immune-deficient SCID mice provides the foundation for human stem cell assays. This review will focus on recent evidence that normal and leukemic human stem cells can be assayed in these systems.     

Process monitoring using auto-associative,feed-forward artificial neural networks

The potential of using artificially simulated neural networks as intelligent, adaptive process-monitoring devices is discussed. The investigation is considered as a method for automatic, intelligent exception reporting for quality control applications. The technique is also compared with the conventional statistical approaches of principal component analysis and Kohonen's feature map. The applications of the technique in aerospace and manufacturing environments are presented and a possible extension of the method to incorporate a diagnostic function is discussed.Seconded from Cheltenham and Gloucester College of Higher Education as a Royal Society/SERC Research Fellow at Smith's Industries Aerospace and Defence Systems, Bishop's Cleeve, Cheltenham, UK.     

Epilepsy, retarded growth and onchocerciasis, in two areas of different endemicity of onchocerciasis in Burundi

A high prevalence of epilepsy (up to 1.3%) was observed in an area hyperendemic for onchocerciasis in the province of Bururi, Burundi. Following this observation, epileptics and controls were examined in 2 communes (administrative units) with different endemicity for onchocerciasis. Altogether, onchocerciasis was more frequent in epileptics (81.8%) than in controls from the same households (68.3%; P < 0.05). This difference was more marked in the hyperendemic area. Other possible causes of epilepsy, including cysticercosis, were infrequent (18 possible cases among 110 epileptics). During this survey, 9 epileptics with growth retardation were seen. They showed most characteristics of Nakalanga syndrome, which was described from Uganda. All 9 such cases were suffering from onchocerciasis. These findings give more evidence of a possible association between onchocerciasis and epilepsy, and between onchocerciasis and Nakalanga syndrome.     

A method for functional magnetic resonance imaging of olfaction

A method for generating olfactory stimuli for humans within a functional magnetic resonance imaging (fMRI) experimental design is described. The system incorporates a nasal-mask in which the change from odorant to no-odorant conditions occurs in less than 500 ms and is not accompanied by visual, auditory, tactile, or thermal cues. The mask provides an ordorant-free environment following prolonged ordorant presence. Specific imaging parameters that are conducive to the study of the human olfactory system are described. In a pilot study performed using these methods, the specific patterns of activation observed converged with published experimental and clinical findings.     

Predictors of underlying coronary artery disease in cocaine associated myocardial infarction: a meta-analysis of case reports

A preliminary report presenting the results of fibular strut grafting in the severely resorbed mandibular and maxillary region is presented. Thirteen patients were treated due to severe resorption of alveolar and basilar bone of 49 segments of the mandible and the maxilla. Two patients additionally had pathological fractures of the mandible. In 10 cases the strut graft was harvested by means of a new minimally invasive technique. After modelling the fibular bone it was fixed to the recipient site by miniscrews or implants. After a mean follow-up period of 20 months (max. 31, min. 11 months) a retrospective analysis of clinical and radiological findings was carried out. It showed that a mean augmentation of 16 mm was achieved. Compared to other studies the fibular strut graft was resorbed less, and due to the primary stability it could be used for the treatment of fractures of the mandible. No more than natural resorption was observed when the patients received their prostheses fixed to dental implants.     

Altered basal and insulin-stimulated phosphotyrosine phosphatase (PTPase) activity in skeletal muscle from NIDDM patients compared with control subjects

To measure possible changes in basal and insulin-stimulated phosphotyrosine phosphatase (PTPase) activity in skeletal muscle from insulin-resistant individuals, soluble and particulate muscle fractions were prepared from biopsies taken before and after a 3-h hyperinsulinaemic euglycaemic clamp in eight non-insulin-dependent diabetic (NIDDM) patients and nine control subjects. We used a sensitive sandwich-immunofluorescence assay and the human insulin receptor as the substrate. PTPase activity was expressed as percentage of dephosphorylation of phosphotyrosyl-residues in immobilized insulin receptors per 2 h incubation time per 83 micrograms and 19 micrograms muscle fraction protein (soluble and particulate fraction, respectively). In the diabetic soluble muscle fractions, the basal PTPase activity was decreased compared with that of control subjects (11.5 +/- 5.5 vs 27.5 +/- 3.3, p < 0.04, mean +/- SEM). In the particulate muscle fractions from the control subjects, PTPase activity was increased after 3 h hyperinsulinaemia (20.0 +/- 3.2 vs 30.2 +/- 3.6, p < 0.03) and in the corresponding soluble fractions PTPase activity seemed decreased (27.5 +/- 3.3 vs 19.9 +/- 5.9, NS). No effect of insulin on PTPase activity was found in NIDDM patients (25.1 +/- 4.1 vs 27.2 +/- 5.2, 11.5 +/- 5.5 vs 15.1 +/- 4.5 [particulate and soluble fractions], NS). In conclusion, we found that the basal PTPase activity in soluble muscle fractions was decreased in NIDDM patients; furthermore, insulin stimulation was unable to increase PTPase activities in the particulate fractions, as opposed to the effect of insulin in control subjects.     

Efficient simulation of hierarchical stochastic activity network models

Stochastic extensions to Petri nets have gained widespread acceptance as a method for describing the dynamic behavior of discrete-event systems. Both simulation and analytic methods have been proposed to solve such models. This paper describes a set of efficient procedures for simulating models that are represented as stochastic activity networks (SANs, a variant of stochastic Petri nets) and composed SAN-based reward models (SBRMs). Composed SBRMs are a hierarchical representation for SANs, in which individual SAN models can be replicated and joined together with other models, in an iterative fashion. The procedures exploit the hierarchical structure and symmetries introduced by the replicate operation in a composed SBRM to reduce the cost of future event list management. The procedures have been implemented as part of a larger performance-dependability modeling package known asUltraSAN, and have been applied to real, large-scale applications. This work was supported in part by the Digital Equipment Corporation Faculty Program: Incentives for Excellence.