High-throughput sample preparation for protein or peptide structural characterization.

High-performance liquid chromatography (HPLC) combined with mass spectrometry has become the method of choice for identifying and characterizing cell-expressed biomolecules. This technology has evolved so rapidly that efficient sample preparation in a high-throughput mode has become a rate-limiting step. Applications using C18 resin, 200 A pore size, and 15-microm bead size silica and sulfonated divinylbenzene particles were tested. The convenient, solvent-resistant, 96-well MultiScreen filter plates with the Multiscreen Column Loader provided efficient removal of salts and detergents and excellent sample recovery for small volumes. The 96-well simultaneous, uniform loading of dry powders, beads, or resins in 45-, 80-, or 100-microL volumes easily accommodates various media capacities and elution volumes. Recovered eluates demonstrated high well-to-well reproducibility during analyte adsorption, washing, and elution. Sample recovery was analyzed by HPLC for a variety of proteins, peptides, and proteolytic digests. The utility of MultiScreen 96-well mini-columns in performing sample clean-up was also demonstrated for HPLC and mass spectroscopy.

MRI-guided biopsy in inflammatory myopathy: initial results.

The purpose of this report is to describe our initial experience with techniques employing magnetic resonance imaging (MRI) to guide the choice of muscle to be biopsied in patients suspected of having inflammatory myopathy. Five patients with a clinical diagnosis of inflammatory myopathy (IM) were studied. Four were imaged prior to biopsy. Four had repeated examinations, either immediately following biopsy or to evaluate disease progression. Use of MRI to localize muscle lesions was associated with abnormal pathologic findings in all cases, including histopathologic demonstration of lymphocyte infiltration in three cases of idiopathic polymyositis; nonspecific myopathic changes were seen in one patient with probable dermatomyositis and in one patient with chronic inflammatory polyneuropathy and high serum creatine kinase levels (> 45,000 IU/ml). The precise location of the area sampled by biopsy was visible in only one of four postbiopsy images. MRI shows promise in identifying pathologic muscle in patients suspected of having one of the inflammatory myopathies; however, further refinement of localization techniques may be needed to optimize histopathologic diagnoses.

Application of the MultiScreen system to cytokine radioreceptor assays.

Radioreceptor assays are becoming increasingly valuable in the biotechnology community for a variety of basic and applied research applications. It is clear, for example, that assessing the potential spectrum of biological activities of a novel polypeptide regulatory factor can be greatly simplified by the development of a rapid radioreceptor assay, since a wide variety of cell types can be screened using a single type of assay. By contrast, searching for potentially diverse biological effects can be an extremely time-consuming process. In addition, screening for agonists/antagonists for hormones using radioreceptor assays has a marked advantage compared with biological assays, in that compounds or natural products that are toxic to cells will not read out as false positives in a binding assay. Our laboratory has developed a major program centered on the molecular characterization of receptors for polypeptide hormones involved in immune regulation, including a number of cytokines/interleukins and also several colony stimulating factors. We have developed a variety of radioreceptor- and fluorescence-based assay systems for ligand-receptor interactions, with applications in basic characterization, purification, cDNA cloning, and drug development screens for cytokine receptors. In this report we compare two assay formats, a standard phthalate oil centrifugation method and a novel plate filtration system, using the interaction between interleukin-1 alpha and its receptor as a test system.

Molecular beam deposition of optical coatings and their characterization.

Significant improvements can be made in the fabrication of optical thin film structures by using molecular beam and ultrahigh vacuum techniques. These lead to the achievement of more stable films and multilayer coatings with improved morphology, density, and resistance to laser-induced damage. The microstructure of the film can be controlled to a high degree by using quasisuperlattice techniques, which also provide a means of refractive index synthesis. This can be applied to simple graded structures or complex periodic gradings as required for Bragglike structures. Etalon filters fabricated using the technique have been used for optical bistability experiments and have exhibited stable operation for periods of many hours under continuous cycling.

Assessment of blood interactions with the Therapore Apheresis System.

Biocompatibility evaluations are a complex issue involving not only classical toxicity and animal testing but also the interactions of biological systems. The evaluations of the Therapore Primary System are reported for three model systems: (1) in vitro human blood, (2) a baboon (Papio anubis) animal model, and (3) normal human volunteer apheresis. Measurements of both complement (C3a) and platelet (BTG and TXB2) activation products during the apheresis procedures are given as a sensitive indicator of biocompatibility. No statistically significant elevations of these activation products were observed. Thus, the Therapore System was found to have negligible perturbations of the interacting biological systems evaluated even with the highly sensitive RIA procedures employed. These observations and methods establish a solid foundation for comprehensive biocompatibility evaluations of future extracorporeal devices.

Anisotropic membranes with carboxypeptidase G1.

Anisotropic polysulfone membranes were prepared with carboxypeptidase G1 embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.