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Biomedical subjects

M Hoare

Publications and source records attributed to M Hoare.

At least 37 records · Page 2Linked to original sources
PubMed1998

Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths.

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.

Crystallization↗
PubMed1996

High speed centrifugal separator for rapid on-line sample clarification in biotechnology.

Sample clarification is a common operation in biochemical analytical methods for removing interfering or unwanted particulates from an analyte sample. Filtration provides one option for removal of particulates. However, in many cases the loss of soluble protein due to filter adsorption is unacceptable and an alternative must be sought. In this paper a microcentrifuge designed to automatically sample, spin, deliver supernatant to an analyser and wash out solids from the bowl is described. The performance of the system is assessed in terms of its clarification efficiency and the time required to achieve satisfactory clarification. Additionally, the effects of different protein precipitating agents on yeast homogenate samples separated using the microcentrifuge are studied where the system is used to deliver supernatant to a flow injection analyser. The paper demonstrates that the microcentrifuge may be used to separate rapidly such samples on a time scale between 10-60 s depending upon the type and size of sample and be successfully used as a component of an at-line monitoring system.

Biotechnology↗
PubMed1996

Analysis of kinetic data of antibody-antigen interaction from an optical biosensor by exponential curve fitting.

An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.

Animals↗
PubMed1996

Posttranslational processing of recombinant human interferon-gamma in animal expression systems.

We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodopera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.

Amino Acid Sequence↗
PubMed1995

Kinetics of protein-protein interactions at the surface of an optical biosensor.

Methods based on the use of optical biosensors have recently become available to provide a convenient means of determining the rate and equilibrium constants for bimolecular interactions between immobilized ligands and soluble ligate molecules. However, the association data that these methods provide are not always accurately described by the expected pseudo-first-order reaction mechanism, particularly when the ligand is immobilized on a dextran matrix. We show that a better description of the association data, especially at higher ligate concentrations, is achieved with a double exponential function, indicating that at least two rate-limiting processes are involved. Various models are considered in order to explain these observations: the presence of two (or more) distinct populations of immobilized ligand; a change, possibly conformational, in the immobilized ligand before or after ligate binding; or the hindrance of ligate binding to immobilized ligand. We suggest that steric hindrance caused by ligate binding to the dextran-coated sensor surface seems the most likely explanation for the observed biphasic association kinetics and that the faster initial phase should be used in oder to determine association constants that can be compared to those in solution.

Antigen-Antibody Reactions↗
PubMed1995

N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems.

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.

Animals↗
PubMed1994

High-resolution separation of recombinant human interferon-gamma glycoforms by micellar electrokinetic capillary chromatography.

Recombinant human interferon-gamma (IFN-gamma) glycoform populations produced by Chinese hamster ovary cells have been resolved by micellar electrokinetic capillary chromatography (MECC). Separations were performed in uncoated fused silica capillaries at alkaline pH in the presence of micellar concentrations of the anionic detergent sodium dodecyl sulfate (SDS). Maximum resolution was obtained reproducibly with high-ionic-strength borate/SDS electrophoresis buffer. Under the conditions described, glycoform migration time was inversely related to the amount of carbohydrate associated with the protein. Digestion of IFN-gamma with peptide-N-glycosidase F allowed virtual real-time monitoring of glycosidase digests by capillary electrophoresis. Analysis of other digestions with either neuraminidase or endoglycosidase H (endo H) showed most IFN-gamma glycoforms to be sialylated and a minor proportion of glycoforms to be associated with oligomannose structures. While both bovine pancreas ribonuclease B and horse-radish peroxidase glycoforms were separated by this technique, proteins glycosylated at multiple sites such as bovine serum fetuin and human alpha 1-acid glycoprotein were not well resolved by MECC.

Amidohydrolases↗
PubMed1994

Conditions for the production of jadomycin B by Streptomyces venezuelae ISP5230: effects of heat shock, ethanol treatment and phage infection.

The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced by Streptomyces venezuelae ISP5230 following a 42 degrees C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient content and concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h after S. venezuelae ISP5230 was inoculated into a D-galactose-L-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replaced L-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increased L-isoleucine and D-galactose concentrations in the production medium.

Anti-Bacterial Agents↗
PubMed1994

Imlink and continuing medical education: the use of an image transfer system to broadcast teaching cases nationally.

Imlink, a personal computer based image storage and transfer system, has been used regularly in Oxford since 1989, primarily to transmit computed tomography (CT) brain scans to the regional neurosurgery centre for management advice. Recently this system has been used to send a weekly educational programme (CT and magnetic resonance imaging neuroradiology cases) to district general hospitals.

Computer Communication Networks↗
PubMed1991

The use of a computerized image transfer system linking a regional neuroradiology centre to its district hospitals.

We describe a new personal computer based image transfer and storage system. We prospectively assessed the uses and benefits of ImLink (Electronic Imaging Ltd.) in a regional neuroradiology department linked by the system to six referring district general hospitals. One hundred and fifty consecutive CT scans transferred by the system were analysed (142 head, four spine and four body scans). Sixty-eight CT scans were of acute neurosurgical cases. A significant change in management occurred in 55 of these cases (81%), principally as a result of prevention of transfer of head-injured patients when no neurosurgically correctable lesion was evident. The high quality of transferred images achieved by the system allows the provision of specialist radiological advice to colleagues in linked district hospitals. A further 50 non-acute head CT scans were transferred by district hospital radiologists seeking the second opinion of a neuroradiologist. Twenty-three out-of-hours acute head CT scans were transferred from clinicians in our own hospital to the home of the on-call neuroradiologist. Overall 63% of the 150 scans were transferred during normal working hours. We discuss the multiple uses and future potential for such an image transfer and storage system.

Adolescent↗
PubMed1990

Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up.

Nucleic acids, lipid, and colloidal particulate material can be selectively flocculated from a yeast cell homogenate by the cationic polymer polyethyleneimine (PEI). Flocculation can occur from a crude homogenate, a homogenate clarified centrifugally, or by the prior use of sodium tetraborate (borax). Flocculation from a homogenate previously clarified by the use of borax is best suited for large-scale operation. The supernatant obtained following centrifugation is effectively free of nucleic acid, lipid, and particulate material with essentially 100% soluble enzyme recovery. Enzyme specific activity increases by approximately 45% compared to a zero PEI control.

Chromatography, Gel↗
PubMed1990

Blunt abdominal aortic trauma.

Due to its well protected position within the abdomen, blunt injuries to the abdominal aorta are uncommon. A review of previous reports in the literature, together with four new cases, are presented here. Motor car accidents are the most common cause of this injury, especially if seat belts are worn. Associated gastrointestinal injuries did not lead to any increased mortality; however, delay in the diagnosis was a significant factor in the deaths of several patients. Prompt recognition and early surgical management are essential in the treatment of this problem.

Accidents, Traffic↗
PubMed1989

Biochemical engineering challenges of purifying useful proteins.

Some of the biochemical engineering issues that arise when the purification of proteins is scaled up are examined. The first question addressed is the quantity of various proteins likely to be required. Consideration is given to the order in which isolation procedures may be applied and to the selective removal on a large scale of non-proteinaceous materials. Some general problems such as the effect of mechanical forces on proteins and their complexes and aggregates are examined, together with ways of enhancing the properties of these aggregates and the methods of recovering them. The manner in which on-line process optimization can be achieved during pilot plant trials is discussed and the paper concludes with a summary of new scientific developments to which the biochemical engineer will need to respond.

Biotechnology↗
PubMed1983

Intermittent sequential pneumatic compression of the legs and thromboembolism-deterrent stockings in the prevention of postoperative deep venous thrombosis.

One hundred fifty patients over the age of 30 who had undergone major abdominal operations were stratified according to the risk of deep venous thrombosis and randomized into three groups to receive different prophylactic regimens: group A, electrical calf stimulation; group B, low-dose subcutaneous heparin; group C, intermittent sequential compression and thromboembolism-deterrent (TED) stockings. All the patients were scanned with the 125I-fibrinogen test for the whole stay in hospital. The incidence of 125I-fibrinogen detected deep venous thrombi was 18% in group A, 9% in group B, and 4% in group C. The results indicate that the regimen of intermittent sequential compression and TED stockings is as effective as low-dose subcutaneous heparin. Electrical calf stimulation is less effective.

Abdomen↗
PubMed1982

The effect of local cooling on digital systolic pressure in patients with Raynaud's syndrome.

The effect of local controlled cooling on the digital systolic blood pressure in the hand was studied in 25 healthy volunteers and 25 patients with Raynaud's syndrome. Arterial systolic pressure was indirectly measured using a digital cuff over the proximal phalanx and a photoplethysmographic probe over the digital pulp to detect pulsation. Pressures were measured after occlusion of the circulation (cuff at 180 mmHg) for 5 minutes with water circulating initially at 30 degrees C and then at 10 degrees C. An air filled cuff was used simultaneously on a control finger. The digital systolic pressure at 10 degrees C in the patients was significantly lower (P less than 0.001 Mann-Whitney U test) than in the normal volunteers whilst calculation of the percentage change in pressure induced by cooling the digit to 10 degrees C allowed complete separation between both groups. The range in the controls was +2 per cent to -26 per cent and in the patients, -33 per cent to -100 per cent. The test gives reproducible results (coefficient of variation 5-11 per cent) and offers an objective method of identifying patients with excessive vasospasm due to a cold stimulus. It may prove useful in studying natural history and the effect of drug therapy.

Adult↗