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.     

Estimation of deformation gradient and strain from cine-PC velocity data

Phase contrast magnetic resonance imaging (MRI) can provide in vivo myocardial velocity field measurements. These data allow densely spaced material points to be tracked throughout the whole heart cycle using, for example, the Fourier tracking algorithm. To process the tracking results for myocardial deformation and strain quantification, we developed a method that is based on fitting the tracking results to an appropriate local deformation model. We further analyzed the accuracy and precision of the method and provided performance predictions for several local models. In order to validate the method and the theoretical performance analysis, we conducted controlled computer simulations and a phantom study. The results agreed well with expectations. Human heart data were also acquired and analyzed, and provided encouraging results. At the signal-to-noise ratio (SNR) level and spatial resolution expected in clinical settings, the study predicts strain quantification accuracy and precision that may allow the technique to become a practical and powerful noninvasive approach for the study of cardiac function, although clinically acceptable data acquisition strategies for three-dimensional (3-D) data are still a challenge.     

Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF-alpha) gene promoter and with elevated TNF-alpha levels in tear fluid

Tumor necrosis factor alpha (TNF-alpha) may play a central role in the disease pathogenesis which occurs as a consequence of chlamydial infection. To investigate the importance of TNF-alpha gene promoter polymorphisms and TNF-alpha levels in tear fluid in scarring trachoma, a large matched-pair case-control study was performed in The Gambia. The -308A allele was present in a higher proportion of patients (28.4%) than controls (18.4%), with an increasing association for homozygotes (chi2 for trend, P = 0.032; allele frequency, 0.163 in patients and 0.099 in controls; chi2, P = 0.025). For the -238A allele, the association was similar but not significant. The disease association was highly significant when the number of either -308A or -238A sites in an individual was considered (P = 0.003). TNF-alpha promoter alleles are tightly linked to some HLA class I and II alleles, but multivariate analysis confirmed that the disease associations were independent of HLA, although a class I allele, A*6802, is also associated with disease. TNF-alpha was more frequently detected in tear samples from patients (27.6%) than from controls (15.9%), increasingly so for higher levels of detectable TNF-alpha (P = 0.015). Among patients, detectable TNF-alpha in tears was highly associated with the presence of ocular chlamydial infection (P < 0.001). The results indicate that TNF-alpha plays a major role in the tissue damage and scarring which occurs as a consequence of Chlamydia trachomatis infection.     

Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle

Attempts to immunise sheep against natural infestations by Lucilia cuprina larvae have not been effective. Yet it is known that the larvae excrete the immunosuppressant ammonium bicarbonate. The effect of larval ammonium and nonionic ammonia on immunopathobiology was evaluated in 12 infested sheep. The concentration of ammonium in veins draining infested sites was measured in another group of four sheep. Mean jugular unionized ammonia concentration increased 3.5 to 5.6 times above pre-infested control levels. Mean venous ammonium concentrations draining infested sites were 13 times higher than pre-infested jugular or carotid levels. Increases in jugular ammonia concentrations correlated with increased number of larvae, area of infestation, earlier death, neutropenia, eosinopenia, lymphocytopenia, large declines in serum globulins and zinc, and large rises in toxic neutrophils. The high concentrations of toxic unionized ammonia in blood directly permanently damaged neutrophils and lymphocytes and depressed serum globulin production. The results show that the ammonium from the excreta of larvae of L. cuprina may be highly immunosuppressive.     

A delta transform approach to loop gain-phase shaping design of robust digital control systems

This paper addresses the existence of loop gain-phase shaping (LGPS) solutions for the design of robust digital control systems for SISO, minimum-phase, continuous-time processes with parametric uncertainty. We develop the frequency response properties of LGPS for discrete-time systems using the Δ-transform, a transform method that applies to both continuous-time and discrete-time systems. A theorem is presented which demonstrates that for reasonable specifications there always exists a sampling period such that the robust digital control problem has a solution. Finally, we offer a procedure for estimating the maximum feasible sampling period for LGPS solutions to robust digital control problems.     

Hand, space and attentional asymmetries in goal-directed manual aiming

Two experiments were conducted to explore the interaction of the two cerebral hemispheres in motor control, by examining hand, space and attentional asymmetries in goal-directed aiming. In Experiment 1, right-handed subjects moved to targets more quickly with their right hand than their left hand. In addition, each hand was faster when moving in its own hemispace. Although in a control condition, movements were initiated more quickly with the left hand, visual distractors disrupted left hand performance more than right hand performance. For contralateral aiming, ipsilateral distractors caused the greatest interference. In Experiment 2, when targets and distractors were all presented at the midline, a right hand advantage was found for movement time along with a left hand advantage for reaction time, independent of target and distractor location. Our findings are discussed in terms of a right hemisphere role in movement preparation and the allocation of attention in space, and greater left hemisphere involvement in movement execution.