Food proteins from yeast-based precision fermentation: Simple purification of recombinant ß-lactoglobulin using polyphosphate.

Proteins produced through precision fermentation are often purified through chromatographic methods. Faster and more cost-effective purification methods are desired for food application. Here, we present a simple method for purification of protein produced from yeast, using ß-lactoglobulin secreted from Pichia pastoris as an example. The food-grade salt hexametaphosphate (HMP) was used to precipitate the protein at acidic pH, while the impurities (extracellular polysaccharides; mainly mannan) remained soluble. After re-solubilization of the protein-HMP complex by neutralization, excess HMP was selectively precipitated using calcium chloride. The protein content of the crude sample increased from 26 to 72 wt% (comparable to purification with anion exchange chromatography), containing only residual extracellular polysaccharides (9 wt%) and HMP (1 wt%). The established method had no significant impact on the structural and functional properties (i.e., ability to form emulsions) of the protein. The presented method shows potential for cost-effective purification of recombinant proteins produced through yeast-based expression systems.

Determination of specific and non-specific protein-protein interactions for beta-lactoglobulin by analytical ultracentrifugation and membrane osmometry experiments.

Protein-protein interactions are essential for the understanding of biological processes. Specific protein aggregation is an important aspect for many biological systems. In particular, electrostatic interactions play the key role for protein-protein interactions, as many amino acids have pH-dependent charge states. Moreover, protein dissociation is directly related to the solution pH, ionic strength, temperature and protein concentration. The subtle interplay between different specific and non-specific interactions is demonstrated for beta-lactoglobulin (BLG) with a focus on low salt concentrations, thus mimicking technically relevant processing conditions. BLG is a well-characterized model system, proven to attain its monomer-dimer equilibrium strongly dependent upon the pH of the solution. In this manuscript, we present a unique combination of analytical ultracentrifugation and membrane osmometry experiments, which quantifies specific and non-specific interactions, i.e. in terms of the dimer dissociation constants and the second osmotic virial coefficient, at pH 3 and 7 and sodium chloride concentrations of 10 mM and 100 mM. This provides direct insight to protein-protein interactions for a system with a concentration-dependent monomer-dimer equilibrium. Moreover, using a coarse-grained extended DLVO model in combination with molecular dynamics simulations, we quantify non-specific monomer-monomer, monomer-dimer and dimer-dimer interactions as well as the binding free energy of BLG dimerization from theoretical calculations. The experimentally determined interactions are shown to be mainly governed by electrostatic interactions and further agree with free energy calculations. Our experimental protocol aims to determine non-specific and specific interactions for a dynamically interacting system and provides an understanding of protein-protein interactions for BLG at low salt concentrations.

Comparison of nitrergic signaling in circular and longitudinal smooth muscle of murine ileum.

BACKGROUND: Gastrointestinal (GI) motility originates from coordinated movements of circular (CM) and longitudinal (LM) smooth muscle. How the two muscle layers react individually to nitrergic input and how they integrate nitrergic signaling is not thoroughly understood. METHODS: We used immunohistochemistry to unveil expression of NO-sensitive guanylyl cyclase (NO-GC) in the ileum. For functional analyses, we measured tone of ileal CM and spontaneous contractions in both ileal muscle layers from mice lacking NO-GC globally (GCKO) and specifically in smooth muscle cells (SMC-GCKO). KEY RESULTS: In contrast to other parts of the GI tract, NO-GC was not expressed in ckit-positive cells in ileum. NO-GC expression was intense in platelet-derived growth factor receptor α-positive cells and in yet unidentified cells of myenteric plexus and serosa. Both CM and LM developed spontaneous contractile activity; frequency and duration of their spontaneous contractions were identical. The amplitude of spontaneous contractions in CM was increased in the absence of NO-GC. In ileum from control (ctrl) animals, inhibition of NO-GC increased whereas NO-GC stimulation decreased tissue tone. In contrast, contractile activity in LM was not different between ctrl and knockout strains. Here, NO led to suppression of spontaneous contractions of ctrl ileum whereas GCKO tissue was unaffected. To our surprise, NO suppressed spontaneous contractions in SMC-GCKO ileum indicating participation of other cell type(s). CONCLUSIONS AND INFERENCES: NO-GC in SMC is involved in the regulation of tone and amplitude of spontaneous contractions in ileal CM. In LM, NO induces suppression of spontaneous contractions via NO-GC in a non-SMC type.

A cost analysis of positron emission tomography.

OBJECTIVE: Changes in regulations and improvements in reimbursement have propelled positron emission tomography (PET) into clinical use, making it increasingly important to understand the costs of this emerging service. Cost analyses are important tools to do this. Data published previously on these topics reflect assumptions that are no longer valid. The aim of this study was to determine the cost of developing and operating a PET facility and to evaluate whether a regional cyclotron serving several scanners reduces costs. MATERIALS AND METHODS: Financial data were collected on capital expense and global operating costs through interviews with industry experts, evaluation of prior studies, and review of expenses incurred at the University of Southern California PET center. A data model and cost templates were developed. Expenses were allocated either to the production or purchase of radiopharmaceuticals or to the provision of the PET scan, and the cost per procedure was determined. A sensitivity analysis was performed on the net present value for key parameters. RESULTS: A cyclotron serving a single scanner is not financially viable. The radiopharmaceutical distribution configurations were financially sound. In these cases, the cost of the radiopharmaceutical was approximately $700 per dose with modest levels of production (12 doses per day). In addition, the average cost of PET scans (technical scan and professional charges) ranged from approximately $900 to $1400. The critical factor for profitability was shown to be throughput. CONCLUSION: This analysis provides significant insight into the cost of PET and the comparative costs of offering PET through four operating configurations. Reductions in equipment prices, increased availability of radiopharmaceuticals, growth in demand, and improvements in reimbursement have all contributed to the financial viability of this imaging technique.

Federal regulations and reimbursement for PET.

OBJECTIVE: The regulatory and reimbursement environment for PET has changed significantly over the past several years. The Food and Drug Administration's (FDA) findings of the safety and efficacy of key PET drugs have been published, as well as guidelines for the applications to produce PET drugs. In addition, the national Medicare coverage policy for PET has been expanded, most recently with additional indications and coverage restrictions added as of July 2001. The payment rates under the new Hospital Outpatient Prospective Payment System (HOPPS) have been set for PET as well. This communication reviews these recent changes and discusses their impact on the development and operation of a PET center. After reading this article, the nuclear medicine technologist should be able to: (a) state the indications for the use of PET drugs that have been found to be safe and effective by the FDA; (b) detail the general procedures a PET drug production site would have to undertake to be in compliance with FDA regulations; (c) list specific studies that have been approved for payment by Medicare; and (d) describe billing codes used for PET scans. Clarification of regulatory and reimbursement issues is leading to rapid expansion of clinical PET. Keeping abreast of these changes will ensure the successful expansion of any nuclear medicine program to include PET services.

The current status of positron emission tomography.

Positron emission tomography (PET), invented over 25 years ago, is the only imaging technique that provides images of the biological basis of disease. Since disease is a biological process, PET routinely detects disease when other imaging studies, such as CT and MRI, are normal. In addition to its clinical effectiveness, PET has been shown to reduce costs, primarily due to the elimination of other less accurate diagnostic tests and ineffective surgeries. PET has been determined to be applicable to a number of specific applications in the areas of: imaging cancer patients, characterizing myocardial blood flow and viability, and brain imaging in various physiological and pathologic conditions. Tremendous progress has been made in resolving the regulatory and reimbursement issues facing the field of PET. Working with HCFA, representatives of the Institute for Clinical PET and the Society of Nuclear Medicine have brought about expanded HCFA coverage for PET. When HCFA first authorized payment for PET, all coverage decisions were restricted to HCFA and an expanded national coverage policy. HCFA revised its national coverage policy in 1997; this was the first of several steps taken by HCFA towards careful expansion of PET reimbursement. In March 1999, three new indications for whole-body PET scans were added to Medicare's coverage policy. The Institute for Clinical PET is continuing to work with HCFA on continued, appropriate expansion of the coverage policy. This article is partially excerpted from a written statement made by Terry Douglass, Ph.D., president of CTI, Inc., on May 12, 1999, before the Senate Committee on Commerce, Science and Transportation and its Subcommittee on Science, Technology and Space. This was part of the committee's study of "Emerging Technologies in the New Millennium."

PET and [18F]-FDG in oncology: a clinical update.

Positron emission tomography (PET) has become a very useful adjunct to anatomic imaging techniques, adding unique information to the characterization of disease. The whole-body PET FDG technique developed over the last few years has surpassed most expectations with respect to its utility in clinical oncology. The large spectrum of neoplasms that now can be studied with this approach makes it an essential clinical imaging tool in diagnosis and management for many patients with cancer. The metabolic information provided by this technique is complementary to results from standard clinical and morphological examinations. It may be anticipated that through application of the multi-modality imaging approach, significant advances in medical care will come.

Positron emission tomography: a financial and operational analysis.

Positron emission tomography (PET) is an emerging clinical imaging technique that is facing the challenges of expansion in a period of imminent health care contraction and reform. Although PET began showing utility in clinical medicine in the mid-1980s [1], its proliferation into mainstream medical practice has not matched that of other new imaging technologies such as MR imaging. Many factors have contributed to this, including the changing health care economy, the high cost of PET, the length of time it takes to develop a PET facility, and its inherent complexity. In part because of the proliferation of the use of other technologies and the general explosion of costs, insurance carriers are now holding diagnostic techniques, including PET, to stricter standards of efficacy. New techniques must show improvement in long-term outcome of patients, a difficult task for diagnostic tools. In addition to these issues, PET is an expensive technology that requires highly trained multidisciplinary personnel. Questions have also been raised about the most appropriate mechanism for regulation of PET isotope preparation, leading to speculation about future regulatory requirements. The current pioneers of PET must meet these challenges in order for it to become a routine imaging technique. Because of its clinical value, PET will probably survive despite the challenges. For many reasons, though, not every hospital should necessarily develop PET services. Conversely, many hospitals without this technology should consider acquiring PET. The purpose of this article is to identify the financial, operational, and clinical challenges facing PET centers today, describe potential organizational configurations that may enable PET to survive in an antitechnology environment, and delineate which institutions should consider this new technology.

Rehabilitation in spinal cord injury.

Caring for the new quadriplegic is an immense undertaking involving multiple health care professionals who must function as a team. The physician in charge must understand rehabilitation principles and be able to communicate with all members of the team caring for the patient. Rehabilitation principles, if considered early, can greatly decrease the length of stay of the patient and allow him to maximize his potential for recovery. A basic knowledge of physicial and occupational therapy in paraplegia and quadriplegia in the acute stage as well as functional expectations in the long term have been presented.

Diabetic Charcot spine as cauda equina syndrome: an unusual presentation.

Some 6% to 21% of Charcot joints occur in the spine. The underlying disease is usually tabes dorsalis, but diabetes mellitus is another etiology. Degeneration of spinal elements is accelerated and lumbar spinal stenosis with weakness may occur, as has been reported in tabetic arthropathy. The case presented is unusual in two respects: first, the Charcot spine was secondary to diabetic complications, which resulted in a compressive cauda equina syndrome; second, the patient presented with progressive paraparesis and bowel and bladder dysfunction but physical examination by several examiners revealed no clinically evident sensory abnormality. The patient had vague and inconsistent sensory complaints for several years preceding definitive workup, but the overall picture of his disease process only could be made following multiple laboratory, electrodiagnostic, microbiologic, and radiologic testing. The patient presented with subacute paraparesis, providing a wide differential diagnosis ranging from Guillain-Barré syndrome to spinal neoplasm. The physical, radiologic, laboratory, electrophysiologic, histologic/pathologic findings, treatment, and recovery status are included in this report.