ABSTRACT
In this article, the new Dutch pediatric guideline Brief Resolved Unexplained Event is discussed, which replaces the old guideline Apparent Life Threatening Event. The main goal of the new guideline is identification of a group of low-risk infants who need not be admitted to the hospital and in which only limited diagnostic workup is indicated. Three fictional cases are presented to highlight the major changes in management of infants who present with an unexplained event. Application of the new guideline will likely result in less clinical admissions and diagnostic testing in these patients.
Subject(s)
Brief, Resolved, Unexplained Event , Infant, Newborn, Diseases , Sudden Infant Death , Infant, Newborn , Infant , Humans , Child , Risk Factors , HospitalizationABSTRACT
Recovery of recombinant proteins from endogenous, host molecules can be an experimentally intensive and time-consuming task. Often the time to analyze material during development of recovery procedures is the rate-limiting step. Nowadays, modern techniques and equipment are being specifically engineered to make this effort much more efficient. We present a case study to illustrate how a new, automation tool, designed for easy, systematic methods development, can be used for very rapid process and analytical optimization. This tool uses robotics to integrate process development with rapid LC-based analysis requiring no user intervention. The methods and procedures described can be generalized to any recombinant protein recovery campaign.
Subject(s)
Chromatography, High Pressure Liquid/methods , Recombinant Proteins/isolation & purification , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Immunoglobulin G , Robotics , Staphylococcal Protein A/isolation & purificationABSTRACT
Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.
Subject(s)
Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Vasoactive Intestinal Peptide/isolation & purification , Amino Acid Sequence , Chromatography, High Pressure Liquid/methods , Molecular Sequence DataABSTRACT
An extremely rapid assay technique for antibodies has been developed utilizing protein A or protein G bound to Perfusion Chromatography support matrices. Either dilute or concentrated samples are directly injected on a column that selectively binds antibody, which is quantitated directly by elution and UV absorbance. Due to the unique mass transport characteristics of the supports, total assay cycle times are typically 1 minute or less, with assays as short as 15 seconds possible. The assay system can accurately quantitate a 100,000:1 or greater dynamic range in sample concentration without sample dilution, is extremely repeatable and is easy to automate with conventional HPLC systems. Assay of antibodies in a wide range of sample types has been demonstrated.
Subject(s)
Chromatography, Affinity/methods , Immunoglobulin G/analysis , Animals , Calibration , Humans , Ligands , Mice , Nerve Tissue Proteins , Reproducibility of Results , Sensitivity and Specificity , Staphylococcal Protein AABSTRACT
The isoelectric point distribution of G-3-P DH and TPI from human lenses was examined as a function of age and cataract formation. Both enzymes exhibited progressive heterogeneity with age and a shift towards an acidic charge. Little qualitative differences in the pI profiles of G-3-P DH and TPI were found to distinguish mixed cataracts from age comparable normal lenses. While the most alkaline form of G-3-P DH required less HAsO4= for optimal activity, no other kinetic property, i.e. Km substrate, cofactor and inhibitors distinguished any of the charge forms of G-3-P DH. All metaor isozyme forms of TPI had the same Km substrate in the forward and reverse reaction direction. The most acidic forms of G-3-P DH and TPI were less stable to increased temperatures than their more alkaline counterparts suggesting a decreased stability.
Subject(s)
Carbohydrate Epimerases/metabolism , Cataract/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Lens, Crystalline/growth & development , Triose-Phosphate Isomerase/metabolism , Adolescent , Adult , Aging , Child , Female , Fetus , Humans , Infant , Isoelectric Focusing , Lens, Crystalline/enzymology , Middle Aged , Pregnancy , ThermodynamicsABSTRACT
Glyceraldehyde-3-phosphate dehydrogenase has been shown to occur in three different forms in the human adult cataractous lens: a membrane bound form (M) and at least two cytosolic isozymes: I1 and I2. Similar Km's for substrate, cofactor and HAsO4 were established for each form and all three forms, to differing degree, require a reduced sulfhydryl group for maximum activity. A variety of phosphonucleosides (ATP, ADP, AMP and 3' 5' cyclic AMP) as well as NADH inhibit enzyme activity. Inhibition by ATP is non-competitive whereas cyclic AMP and NADH compete for the cofactor binding site. Chloride ion stimulates and inhibits enzyme activity at low and high concentrations respectively.