Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.     

Bacteremia and sepsis

Recent evolution in the thinking of sepsis syndrome has provided a framework on which new clinical and basic research can be built. The separation of the inciting event and the cascade of subsequent physiologic changes has profound effects on how sepsis is thought of and ultimately how it will be treated. Early identification and treatment of infections and identifying patients at risk, to prevent SIRS, is the current role of Eps. Resuscitation of severe sepsis is more complex than other forms of shock and may require extensive resources if rapid admission or transfer to an intensive care unit is not available. As in many instances, the EP must be knowledgeable and skilled in the early identification and initial management of these patients until the definitive care can be provided. Modulation of the inflammatory response appears to be a prime prospect, but its practicality remains to be proved. Research and future roles of EPs include defining the population of ED patients at risk for SIRS and use of mediators of the inflammatory response. Emergency medicine is positioned in a critical point in the care of these patients.     

Clinical relevance of calreticulin in systemic lupus erythematosus

Calreticulin is an abundant intracellular protein which is proposed to have numerous biological functions. However, there is increasing evidence to suggest that calreticulin plays a multifunctional role as an autoantigen present in patients with systemic lupus erythematosus. In this review we detail some of the recent evidence which indicate that calreticulin may play a supportive role in the formation of the autoantigen complex-Ro/SS-A. In addition, several proposed mechanisms of release and surface expression of calreticulin are described in relation to SLE mediated responses to the autoantigen. In particular, the generation of autoantibodies to specific regions of the protein and the ability of calreticulin to interfere with complement mediated inflammatory processes.     

Serotonin function and treatment response to clozapine in schizophrenic patients

OBJECTIVE: Clozapine is the only compound proven to be effective in the 20% of schizophrenic patients refractory to treatment with conventional neuroleptics. Although its mechanism of action has not been elucidated, clozapine appears, in contrast to most conventional neuroleptics, to be a potent serotonin (5-HT) antagonist. This study hypothesized that 5-HT function is increased in patients who benefit from clozapine treatment relative to patients who fail to improve on it. METHOD: The 5-HT receptor agonist m-chlorophenylpiperazine (MCPP) was used as a probe to examine 5-HT function. MCPP (0.35 mg/kg p.o.) was administered in a placebo-controlled design after a 3-week drug-free period to 19 schizophrenic patients. ACTH, prolactin, body temperature, behavior, and MCPP blood level were measured. Patients were then treated with a conventional neuroleptic, and, having failed to respond to it, were treated with clozapine for 5 weeks (up to 600 mg/day). RESULTS: Patients who responded to clozapine had significantly higher ACTH responses to MCPP during the drug-free state than the patients who failed to benefit from clozapine. Moreover, the degree of improvement with clozapine, particularly the improvement in psychotic symptoms, was strongly correlated with the magnitude of MCPP-induced ACTH release. Other MCPP-induced responses and MCPP blood level were similar for the two groups and did not correlate with the degree of symptomatic improvement with clozapine. CONCLUSIONS: Results of this study suggest that MCPP-induced ACTH release, and by inference 5-HT receptor function, may be increased in patients who benefit from treatment with clozapine relative to patients who fail to improve on this drug.     

Differentiation of Peats Used in the Preparation of Malt for Scotch Whisky Production Using Fourier Transform Infrared Spectroscopy

It is not known if peats derived from different areas of Scotland have distinctive chemical constituents that could impact on malt whisky in discernible organoleptic ways. Fourier transform‐infrared (FT‐IR) spectroscopy was used as a high throughput screening method to investigate discrimination of a large number of peat samples from six different geographical origins around Scotland. The data were analysed statistically (using principal component‐discriminant function analysis) and the results showed a difference between peat samples from different geographical origins. Therefore, we have shown that FT‐IR spectroscopy provides a quick and simple method for differentiating peat types.     

The role of laparoscopy in the management of suspected pancreatic and periampullary malignancies

Laparoscopic evaluation of patients with suspected periampullary malignancies has been utilized more frequently in recent years. Its exact role with regard to staging and surgical bypass for palliation have yet to be clearly defined. To better define the role of laparoscopy in the evaluation and palliation of periampullary malignancy, a retrospective review of the Duke experience was carried out. Fifty-three patients with suspected pancreatic or periampullary malignancies were referred for surgical evaluation at Duke University Medical Center between 1993 and 1995. All patients underwent CT scanning and lesions were classified as resectable or unresectable based on previously established criteria. Patients either underwent laparoscopic evaluation (n = 30; 11 with laparoscopic palliation) or proceeded directly to celiotomy (n = 23). Charts were reviewed for postoperative course including complications, length of stay, and hospital costs. Although laparoscopy had a sensitivity of 93.3% for metastatic disease, CT scans accurately staged 86.8% of patients missing only one patient with peritoneal/hepatic disease. Based on these results, laparoscopy may not be beneficial for every patient with a suspected pancreatic malignancy. Retrospectively an attempt was made to determine which patients benefited from laparoscopy and which patients are best served by proceeding directly to open exploration. From these data we devised an algorithm that outlines an efficient and cost-effective approach for this patient population.     

Go green-a sound business decision (part 1)

Speakers at the 1996 Midlands Region Symposium (Go green - like it or lump it) ranged from dyemakers to retailers; Rebecca Reid reports on their predictions for the industry.     

Stereo perception and dead reckoning for a prototype lunar rover

This paper describes practical, effective approaches to stereo perception and dead reckoning, and presents results from systems implemented for a prototype lunar rover operating in natural, outdoor environments.The stereo perception hardware includes a binocular head mounted on a motion-averaging mast. This head provides images to a normalized correlation matcher, that intelligently selects what part of the image to process (saving time), and subsamples the images (again saving time) without subsampling disparities (which would reduce accuracy). The implementation has operated successfully during long-duration field exercises, processing streams of thousands of images.The dead reckoning approach employs encoders, inclinometers, a compass, and a turn-rate sensor to maintain the position and orientation of the rover as it traverses. The approach integrates classical odometry with inertial guidance. The implementation succeeds in the face of significant sensor noise by virtue of sensor modelling, plus extensive filtering.The stereo and dead reckoning components are used by an obstacle avoidance planner that projects a finite number of arcs through the terrain map, and evaluates the traversability of each arc to choose a travel direction that is safe and effective. With these components integrated into a complete navigation system, a prototype rover has traversed over 1 km in lunar-like environments.