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Biomedical subjects

C J Roberts

Publications and source records attributed to C J Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1997

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.

Antiviral Agents↗
PubMed1997

Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces.

The adsorption of a range of plasma proteins to metal and polymer surfaces has been examined using surface plasmon resonance (SPR). The adsorption of proteins was initially studied on the SPR silver sensor surface, and then on a model polystyrene film spun coated directly onto this substrate. In both cases, reproducible adsorption profiles for albumin were attained which compared well with corresponding atomic force microscopy (AFM) and ellipsometry data on protein monolayer packing and thickness respectively. The SPR data revealed the influence of concentration on both protein adsorption kinetics and the time for formation of a monolayer coating. SPR data also highlighted different adsorption kinetics and final monolayer SPR angle shift values for three plasma proteins which have been interpreted in terms of their molecular dimensions and orientation at the polymer interface. AFM data confirmed the presence of a closely packed protein layer for all three protein systems. These studies are discussed in terms of employing SPR in the study of protein interactions at surfaces which are important in the design and evaluation of novel biomedical polymeric materials.

Adsorption↗
PubMed1997

Atomic force microscopic study of the surface morphology of apatite films deposited by pulsed laser ablation.

Atomic force microscopy (AFM) has been used to study the surface morphology of apatite films deposited on metallic and polyethylene substrates by laser ablation using KrF and transversely excited atmospheric CO2 lasers. The films are found to consist of a smooth apatite coating with macroparticles scattered on the surface. A wide variety of macroparticles, differing in size, shape and roughness, were found and analysed employing the high spatial resolution of AFM (< 1 nm). We have investigated the correlation between the apatite film morphology and the deposition conditions. Of particular importance are laser fluence, gas pressure, the nature of the target and the substrate temperature. We have explained these dependencies on the basis of a theoretical model which includes evaporation and a cluster-type laser ablation mechanism.

Apatites↗
PubMed1997

Ocular pain: a casualty study. The spectrum and prevalence of pain in acute eye disease.

This study examined the prevalence and severity of ocular pain in eye casualty patients. All new patients presenting over a 1 month period were invited to indicate their pain level using a visual analogue scale. The results for 352 patients were analysed, and median pain levels calculated for 29 common diagnoses. Of those responding, 94% (= 47% of all new patients) had ocular pain at presentation. While many results were predictable, some diagnoses were associated with higher pain scores than expected. Junior ophthalmologists also were asked to indicate their perceptions of pain severity for the 29 diagnoses using the same visual analogue scale.

Acute Disease↗
PubMed1997

Data analysis using the Internet: the World Wide Web scanning probe microscopy data analysis system.

The first interactive world-wide web-based image analysis system is presented (http://pharm6.pharm.nottingham.ac.uk/processing/main. html). The system, currently tailored to scanning probe microscopy image data, has been developed to permit the use of software algorithms developed within our laboratory by researchers throughout the world. The implementation and functionality of the scanning probe microscopy server is described. Feedback from users of the facility has demonstrated its value within the research community, and highlighted key operational issues which are to be addressed. A future role of Internet-based data processing software is also discussed.

Algorithms↗
PubMed1997

Effect of hormones on the variation of radiosensitivity in females as measured by induction of chromosomal aberrations.

The frequency of dicentrics + ring (dic/cell) and total chromosome aberrations (dicentrics, rings and excess acentrics, etc.) per cell (TAb/cell) has been studied in 50 male and female volunteers after high or low dose rate (HDR, LDR) irradiation of peripheral blood lymphocytes. The mean male aberration frequencies per cell after HDR irradiation were 0.38 dic/cell and 0.61 TAb/cell; following LDR irradiation, the mean aberration frequencies were 0.28 dic/cell and 0.45 TAb/cell. Equivalent female values after HDR irradiation were 0.42 dic/cell and 0.71 TAb/cell; after LDR irradiation, the mean aberration frequencies were 0.30 dic/cell and 0.48 TAb/cell. Analysis of variance showed that there was a highly significant difference between males and females have a greater HDR, but not LDR, irradiation It is concluded from this study that females have a greater variability in their radioresponse, and that this variability is related to progesterone, which has a profound effect upon radiosensitivity, as measured by cytogenetic end points.

Adult↗
PubMed1996

In situ observation of streptavidin-biotin binding on an immunoassay well surface using an atomic force microscope.

Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.

Bacterial Proteins↗
PubMed1996

A novel organic solvent-based coupling method for the preparation of covalently immobilized proteins on gold.

A novel organic solvent-based coupling method has been developed for the covalent immobilization of biological material to gold surfaces. The method employs the polar organic solvent anhydrous 2,2,2-trifluoroethanol as the reaction medium and involves dissolution of the protein (catalase) in the solvent allowing protein coupling to proceed under basic conditions in a dry organic environment. The advantage of this method is that protein attachment is favored over hydrolysis of the coupling reagent. We have shown qualitatively and quantitatively that following attachment to the gold surface a significant proportion of the enzyme catalase remains catalytically active (at least 20-31%).

Gold↗
PubMed1996

The effects of acute phosphate supplementation in subjects of different aerobic fitness levels.

Six trained cyclists (high-fitness group) and six untrained individuals (low-fitness group), performed a 20-min cycle ergometer exercise test at 70% of maximum oxygen consumption (VO2max) followed by a 30-min rest period and then an incremental ride to exhaustion on two occasions, 1 week apart. Ninety minutes prior to exercise subjects consumed a drink containing either 22.2 g dibasic calcium phosphate (DCP; treatment) or calcium carbonate (placebo). Blood was drawn prior to drink ingestion, during submaximal exercise, during recovery and at exhaustion for determination of blood 2,3-DPG, blood ATP, plasma lactate, plasma phosphate, haemoglobin and haematocrit. Throughout exercise, cardiorespiratory variables [oxygen uptake (VO2), minute ventilation, (VE), respiratory exchange ratio, heart rate and oxygen pulse] were monitored, and ratings of perceived exertion obtained. Although there was a trend for the low-fitness group to have a higher plasma phosphate concentration prior to treatment ingestion, no treatment effects on plasma phosphate were noted at any sample time in either group. 2,3-DPG, VO2, oxygen pulse, VE, time to exhaustion and VO2max were significantly higher in the high-fitness group; however, no differences in these variables were observed as a result of phosphate ingestion. Plasma lactate was significantly lower in the high-fitness group during the submaximal exercise and the recovery period, but again phosphate ingestion had no effect. These results suggest that acute DCP supplementation is not effective as an ergogenic aid and that aerobic fitness level does not affect the response to phosphate supplementation.

2,3-Diphosphoglycerate↗
PubMed1996

Variability in the plasma protein binding of velnacrine (1-hydroxy tacrine hydrochloride). A potential agent for Alzheimer's disease.

OBJECTIVES: To quantify the protein binding of velnacrine in healthy individuals and investigate potential sources of variability. SETTING: Medical School Unit, Southmead Hospital, Bristol. SUBJECTS: Plasma samples were obtained from the following groups: a) 11 healthy volunteers aged 18 to 30 years; b) 10 healthy volunteers aged 73 to 87 years; c) 10 patients aged 65 to 85 years hospitalised for a variety of acute illnesses. METHODS: Aliquots of plasma from the above subjects were incubated with various concentrations of velnacrine, in the presence and absence of tacrine hydrochloride. Standard solutions of human serum albumin and alpha 1 acid glycoprotein were incubated with velnacrine. The degree of protein binding was determined using the Amicon centrifree micropartition system. RESULTS: 1) Over the range of concentrations from 10 to 320 ng.ml-1, there was a decrease in protein binding from 59.1 to 46.7%. 2) At 40 ng.ml-1 the plasma protein binding of velnacrine was 54.8% in the group a subjects, 51.9% in the group b subjects and 53.0% in the group c subjects (NS). 3) The mean total plasma protein concentration was significantly lower in the samples from elderly subjects. The mean albumin and alpha 1 acid glycoprotein concentrations were lower and higher respectively in patients with acute disease. 4) Velnacrine was shown to bind to both albumin and alpha 1-acid glycoprotein, but together they did not account for total binding. 5) The binding of velnacrine was significantly decreased from 59.3 to 43.9% in the presence of a therapeutic concentration (25 ng.ml-1) of THA. There was no evidence that velnacrine displaced THA. CONCLUSION: Protein binding can be discounted as a major source of variation in the relationship between drug concentration and effect.

Acute Disease↗
PubMed1996

The discrimination of IgM and IgG type antibodies and Fab' and F(ab)2 antibody fragments on an industrial substrate using scanning force microscopy.

We have previously employed scanning force microscopy (SFM) to study antibody-antigen molecular interactions on microtiter wells used for enzyme linked immunosorbant assays (ELISA). Here we demonstrate the ability of SFM to image and discriminate different types of antibody and antibody fragments bound to an ELISA well surface. The samples studied include a type IgG antibody with a proportion of bound IgM and two-dimensional films of whole IgG antibody, and Fab' and F(ab)2 antibody fragments. Molecular resolution is achieved in each case despite the size of substrate features exceeding most of the molecular dimensions observed. Analysis of the data shows that the SFM overestimates molecular dimensions by an approximately constant amount, which is proposed to principally result from the effects of a finite probe size and not from deformation of the molecular species due to the imaging forces employed.

Animals↗
PubMed1996

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Endoplasmic Reticulum↗
PubMed1996

The role of scanning probe microscopy in drug delivery research.

The success of a drug delivery system is often dependent on the surface properties of the device. These surface properties will determine the complex dynamic interfacial events that occur when the system is introduced into the aqueous environment of a patient. Development of the scanning probe microscopes has provided a number of very powerful new surface analytical techniques that are making a significant contribution to the characterization of drug delivery systems and the interfacial processes that occur when such systems are exposed to aqueous living environments. In this review, we describe the design and attributes of these instruments and discuss the impact of the techniques on a wide range of drug delivery research. The scanning probe microscopes are providing new insights into important problems concerning drug delivery, including the molecular structure of polymeric biomaterial surfaces, the conformation of target biomolecules, the influence of morphology on biodegradation, the adsorption of proteins to synthetic surfaces, and the structure and interactions of colloidal particles. As the whole field of scanning probe microscopy continues to advance, drug delivery research is set to benefit; in the final section of the review, the future potential derived from the ability to characterize new surface properties under aqueous conditions is discussed.

Drug Delivery Systems↗
PubMed1995

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.

Amino Acids↗
PubMed1995

In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by oestrone-3-O-sulphamate.

Many tumours in endocrine-sensitive tissues, such as the breast and endometrium, are hormone-dependent and the hydrolysis of oestrone sulphate (EIS) to oestrone by oestrone sulphatase (EI-STS) is a major source of oestrogen in such tumours. Oestrone-3-O-sulphamate (EMATE) has been shown to be a potent EI-STS inhibitor in vitro, and in this study its ability to inhibit enzyme activity in vivo was examined. EMATE was initially administered to female rats for 7 days, after which liver EI-STS activity was measured. As EMATE also inhibits a related sulphatase in vitro, dehydroepiandrosterone sulphatase (DHA-STS), its effect on the activity of this enzyme in vivo was also investigated. DHA-STS has a pivotal role in regulating the synthesis of another steroid with potent oestrogenic properties, androstenediol. Administration of EMATE almost completely inhibited liver EI-STS (99%) and DHA-STS (99%) activities and was active when given by the oral or subcutaneous routes. After a single dose of EMATE or following the cessation of multiple doses for 10 days, liver EI-STS activity remained inhibited ( > 95%) for up to 7 and 10 days, respectively. Other compounds, such as 4-hydroxytamoxifen and the "pure" antioestrogen ICI 182,780, which are reported to inhibit EI-STS activity in vitro, did not inhibit activity in vivo. In a preliminary study, EMATE, when injected over a 12-day period, effectively reduced the growth of EIS-stimulated nitrosomethyl-urea-induced mammary tumours in ovariectomised rats and inhibited tumour sulphatase activity in treated animals.

Animals↗
PubMed1995

Scanning tunnelling microscopy studies of beta-amyloid fibril structure and assembly.

Alzheimer's disease is in part characterised by the deposit of beta-amyloid peptide in the form of fibrils in the brain. In this study, the scanning tunnelling microscope (STM) has been used to provide high resolution images of synthetic fibril structure and formation as a function of time. Short fibrils are observed following brief peptide incubation times. At longer incubation periods ribbon like filaments were observed. These results suggest that beta-amyloid self-assembly is an ordered process, with a correlation between time of incubation and length of beta-amyloid filament growth.

Amino Acid Sequence↗
PubMed1995

Development of an ELISA using a universal method of enzyme-labelling drug-specific antibodies. Part I: Detection of dexamethasone in equine urine.

The development, validation, and application of an ELISA for dexamethasone in equine urine is described. The drug-protein conjugate was immobilised in microtitre plate wells and antiserum raised against the same drug-protein conjugate was allowed to compete with sample or standard drug and the immobilised drug-protein conjugate. The proportion of antiserum binding to the immobilised drug-protein conjugate was detected using a biotinylated protein G/extravidin-alkaline phosphatase complex in situ and measurement of the substrate product. The method was used to detect the presence of drug-derived material in unextracted diluted urine after the administration of a single i.m. dose of dexamethasone at approximately 0.04 mg/kg to a thoroughbred horse. Validation of the method was carried out against a radioimmunoassay and GC/MS analysis.

Animals↗