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Biomedical subjects

P R Brown

Publications and source records attributed to P R Brown.

At least 19 recordsLinked to original sources

Separation techniques for biotechnology in the 1990s.

Scientists are constantly looking for better and cheaper separation techniques to replace or complement the current technology. Over the past few decades, and in particular the last 10 years, new separation techniques or modifications of existing techniques have become available for separating compounds from complex sample matrices. There are many areas, however, where the separation technology is not sufficient to achieve high purity and yield while remaining cost effective. In the area of biotechnology, separation techniques are urgently needed to meet demands for ultra-high purity and yield. Thus, a variety of techniques are being developed to address these needs. Generally, biological compounds for the pharmaceutical and biotechnology industries must be obtained at greater than 99.9% purity (sometimes greater than 99.99%) while maintaining high yield. In any area of chemistry this degree of purity would cause problems; in biotechnology it is even more difficult to achieve because of the complex sample matrices. In addition, the compounds of interest may be very similar to impurities or contaminants in the sample matrix, and the compounds could be denatured (or even destroyed) by certain solvents and/or high temperature. In particular, three areas of biotechnology have presented scientists with problems in separations: cell separations, DNA-RNA separations, and protein-peptide separations. The current technology available and possible future trends in these areas are discussed, and also problems to be solved in the future.

Base Sequence

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.

Acquired Immunodeficiency Syndrome

NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme.

Measurements of the deaminating activity of NAD-dependent glutamate dehydrogenase (NAD-GDH) in Pseudomonas aeruginosa strain 8602 (PAC 1) showed an initially constant rate that gave way to a 3.5-fold increased rate on prolonged incubation. Only the faster rate was observed when assay mixtures were preflushed with nitrogen or were treated with the detergent Triton X-100. Comparison of the intracellular distribution of NAD-GDH with marker enzymes showed it to be associated with the cytoplasmic membrane. The results suggest that NAD-GDH may be linked to oxygen through an electron-transport system.

Bacterial Proteins

Dielectric properties of the system bovine albumin: urea: betaine in aqueous solution.

Urea (about 5 mol l-1) causes an approximately five-fold increase of the dielectric increment of bovine serum albumin at 20 degrees C. The increase is reversed by betaine (about 2.5 mol l-1), or prevented if urea and betaine are added together. This result can be seen as an electric counterpart to the protective role of osmolytes which are secreted by cells and tissues subjected to various types of internal or environmental stress.

Betaine

Postcatheterization femoral artery injuries: repair with color flow US guidance and C-clamp assistance.

Color flow ultrasound-guided compression repair of postcatheterization femoral artery injuries was attempted with the assistance of a C-clamp device in 10 patients. The C-clamp device was designed to hold the transducer in the optimal compression position and eliminate operator fatigue. In nine patients, the lesion was eliminated in a mean compression time of 59 minutes. The C-clamp maintained the transducer in an effective compression position in six procedures; in the other four, intervention to prevent slippage was required.

Aged

Chromatographic methods for the analysis of basic neurotransmitters and their acidic metabolites.

Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC-MS and the reversed-phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC-MS is the method of choice. In addition the limits of detection of GC-MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC-MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.

Chromatography, High Pressure Liquid

Evaluation of polyphosphates and polyphosphonates as degradable biomaterials.

A series of polymers, bisphenol A-based poly(phosphoesters), were evaluated as degradable biomaterials. Degradation was observed for the four polymers studied under both in vitro and in vivo conditions. The rate of degradation was affected by polymer side-chain structure and correlated with the swelling behavior. The ethyl side-chain polymers absorbed more water than their phenyl counterparts. Among the sterilization methods, UV irradiation followed by antibiotic treatment was the most suitable, as steam autoclave and ethylene oxide treatments altered the properties of several of the poly (phosphoesters). Tissue response to the poly(phosphoesters) in rabbits was characterized by minor encapsulation and slight or no lymphocyte, giant cell, or macrophage activity. No evidence of edema or necrosis was found. The elastic moduli of these materials varied from 488 MPa for poly(bisphenol A-ethylphosphate) (BPA/EOP) to 627 MPa for the more rigid poly(bisphenol A-phenylphosphonate) (BPA/PP). The ultimate strength, modulus, and energy to failure of BPA/PP were lower than those of similarly compression molded high-molecular-weight poly(L-lactic acid) (PLLA).

Animals

High-performance liquid chromatographic analysis of 3'-azido-3'-deoxythymidine monophosphate diglyceride, an anti-HIV glycerophospholipid.

A reversed-phase chromatographic method is described for the analysis of an experimental anti-AIDS drug 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) [J.M. Steim et al., Biochem. Biophys. Res. Commun. 171, 458-464 (1990)] [1], a phosphatidic acid derivative of AZT. Analytical conditions were based upon conventional separations of glycerophospholipid species. Where AZT-MP-DG was monitored by UV absorption, there were two wavelength maxima. The response was linear in the concentration range used in this study. The peak was characterized by absorbance ratios with a rapid scanning UV detector.

Acquired Immunodeficiency Syndrome

Isolation and purification of lecithin by preparative high-performance liquid chromatography.

Mixed-chain, multispecies, egg yolk-derived lecithin was isolated and purified on a silica column with isocratic elution. A method development column (20 x 0.46 cm I.D.) packed with YMC 15-30 microns, 120 A spherical silica and a mobile phase consisting of 5 mM ammonium acetate in acetonitrile-2-propanol-methanol-water (80:13:5:12) was used to separate the lecithin from other phospholipids. The mobile phase conditions for the method development system was adopted for two types of preparative HPLC systems: a Separations Technology SepTech NovaPrep 5000 system with a 20 x 1.93 cm I.D. column and a ST/800A system with a 20 x 5.00 cm I.D. Annular Expansion (A/E) column. The maximum load was 50 microliters of crude solution (2 mg) for the method development column, 0.90 ml (35 mg) for the 20 x 1.93 cm I.D. column and 6.0 ml (240 mg) for the 20 x 5.00 cm I.D. A/E column. The flow-rates were 2, 35 and 235 ml/min, respectively. The fractions collected from the preparative systems were analyzed for purity by analytical-scale high-performance liquid chromatography and by thin-layer chromatography with selective detection with molybdenum blue for phospholipids and detection of all organic compounds by sulfuric acid. Purity of the recovered lecithin was greater than 99%.

Chromatography, High Pressure Liquid

Use of leeches.

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Animals

Preparative-scale high-performance liquid chromatographic separation and purification of 3'-azido-3'-deoxythymidine-5'-phosphate.

Crude 3'-azido-3'-deoxythymidine-5'-phosphate (AZT-P), obtained from direct phosphorylation of 3'-azido-3'-deoxythymidine (azidothymidine, AZT), was separated and purified by isocratic preparative high-performance liquid chromatography. The components in a 2.5-g load of crude AZT-P, obtained from work-up of the phosphorylation reaction, were separated in 50 min to give 1.8 g of 99.5% pure AZT-P. AZT-P was analyzed by high-performance liquid chromatography and by high-resolution nuclear magnetic resonance (1H, 13C, 31P) spectroscopy. The practical and rapid preparative chromatographic method is being applied to the purification of AZT-P and other antiretroviral dideoxynucleotides, used as intermediates in the synthesis of target-directed experimental drugs for the treatment of AIDS.

Chromatography, High Pressure Liquid

Platelet aggregation by oral streptococci.

One proposed mechanism in the pathogenesis of infective endocarditis is the direct aggregation of platelets by the bacteria causing the disease. Some, but not all, strains of Streptococcus sanguis have been reported to aggregate platelets but the taxonomy of this and related taxa has changed recently. The ability to aggregate platelets by 24 genetically grouped laboratory stock strains was studied along with 8 recent isolates from cases of endocarditis. Strains belonging to S. sanguis could aggregate platelets, but not S. gordonii, "S. parasanguis", S. mitis, S. oralis or related taxa. Also, preliminary data indicate that certain biotypes of S. sanguis lack the ability to aggregate platelets. Of the recent clinical isolates, only 4 aggregated platelets and each of these showed phenotypes typical of S. sanguis. These data suggest that the ability to aggregate platelets is not essential for an organism to be able to cause endocarditis, although it may be a significant virulence factor.

Humans

Reversed-phase liquid chromatographic retention and selectivity surfaces. II. Deoxyribonucleosides.

By plotting capacity factors and selectivity factors as functions of temperature and pH or methanol concentration of the mobile phase, retention and selectivity surfaces of four deoxyribonucleosides were generated. Although changes in the methanol content had little effect on selectivity, changes in pH and temperature could be used to improve selectivity. However, changes in pH or temperature can be coupled with changes in methanol concentration to optimize the analysis time. In addition, by using lines of constant analysis time (isochrons), it was found that several sets of conditions will result in nearly identical retention times. These isochrons can be used to optimize the resolution of the solutes.

Chromatography, Liquid

Analytical-scale high-performance liquid chromatography of omega-3 fatty acid esters derived from fish oils.

Fish oil triglycerides were transesterified to their corresponding methyl and ethyl esters. The esters were separated by reversed-phase high-performance liquid chromatography on a 10-cm column of 5-microns octadecylsilyl silica, using a ternary mobile phase of acetonitrile-tetrahydrofuran-water (9:5:11). The separation of principal saturated and unsaturated C14-C22 fatty acid esters was accomplished in 60 min. For the polyenoic acid esters, detection limits of ca. 26 micrograms were observed, and baseline resolution of the important critical pair of esters of the omega 3 fatty acids, eicosapentaenoic acid (C20:5 omega 3) and docosahexaenoic acid (C22:6 omega 3) was achieved. However, to obtain baseline resolution of the esters from minor fatty acid esters, a urea crystallization procedure was required prior to chromatographic analysis. The influence on the reversed-phase chromatographic retention of four different ester moieties on the fatty acids were also studied. The n-alkyl esters exhibited an increase in ln k' with increasing chain length and with increasing lipophilic character of the alkyl ester moieties; however, with the aromatic benzyl derivatives there was a decrease in ln k' compared with the alkyl esters.

Chromatography, High Pressure Liquid

Dielectric behavior of aqueous solutions of plasmid DNA at microwave frequencies.

The relative permittivity and dielectric loss of aqueous solutions of plasmid (pUC8.c1 and pUC8.c2) DNA have been measured at 20 degrees C over the frequency range 100 MHz-10 GHz. The solutions had a concentration of 0.1% DNA, and were studied both in the relaxed and the supercoiled form. The dielectric measurements were made using a variety of techniques including frequency domain and time domain methods of operation. No evidence of any resonance absorption, nor of any other kind of enhanced absorption, was observed.

DNA, Superhelical