Biomedical subjects

M C McCann

Publications and source records attributed to M C McCann.

At least 19 recordsLinked to original sources

PubMed2000

Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase.

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.

Genetic Engineering↗

PubMed2000

An abundant TIP expressed in mature highly vacuolated cells.

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.

Amino Acid Sequence↗

PubMed1998

A pectate lyase from Zinnia elegans is auxin inducible.

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans-differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell-wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72-h cDNA library with probes from mRNA at time-points of 24 h and 72 h was done revealing a number of transcripts up-regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin-degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N-terminal signal peptide of about 2 kDa, and one potential N-glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium-dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.

Amino Acid Sequence↗

PubMed1998

A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra.

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis. Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.

Arabidopsis↗

PubMed1993

Orientation of macromolecules in the walls of elongating carrot cells.

When round cells from a carrot cell suspension culture are diluted into fresh medium without auxin, the cells elongate to almost 50 times their original diameter within three days. This process of elongation is accompanied by changes in both the composition and the orientation of cell wall polymers. We have obtained information on the orientation of wall polymers in elongating cells by two complementary techniques, one using microscopy and one spectroscopy. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica technique show that walls of round carrot cells have no net orientation of cellulose microfibrils, and that many thin fibres can be seen cross-linking microfibrils. Walls of elongated carrot cells, in contrast, show a marked net orientation of microfibrils at right angles to the axis of elongation. Fourier Transform Infrared (FTIR) spectra obtained from defined areas of single cell walls show that walls of round carrot cells contain more protein, esters and phenolics in a given area (10 microns x 10 microns) than walls of elongated carrot cells, that contain proportionally more carbohydrate. The orientation of particular functional groups, with respect to the direction of elongation of the cell, can be determined by inserting a polariser into the path of the infrared beam, before it passes through a cell wall sample mounted on the stage of the microscope accessory. In the walls of elongated cells, ester bands, amide bands characteristic of proteins, and stretching frequencies in the carbohydrate region of the spectrum all show a net orientation transverse to the long axis of the cells. In the walls of round carrot cells, however, there is no such net orientation of polymers. Spectra obtained from 25 microns-thick fresh sections of the etiolated stem of a carrot seedling show that different wall components are polarised in different tissue types. These techniques have therefore enabled us to define differences in both the composition and the architecture of walls of elongating cells at the level of a single cell, and to suggest that polymers not previously thought to be ordered, such as pectin and protein, are strictly oriented in some wall types.

Antibodies, Monoclonal↗

PubMed1989

Stability of murine monoclonal anti-A, anti-B and anti-A,B ABO grouping reagents and a multi-centre evaluation of their performance in routine use.

We have previously reported the production of 3 murine monoclonal reagents for ABO typing (designated ES-9, ES-4 and ES-15). This study presents results of tests of stability of these 3 reagents, together with a fourth murine monoclonal antibody (LM103/107). In addition, data are also presented from a multi-centre evaluation of the performance of the murine monoclonal reagents in routine ABO typing of both donors and patients using a wide variety of techniques, both manual and automated. The potency and stability of the 4 monoclonal antibody based reagents is compared with a broad selection of monoclonal and polyclonal ABO typing reagents. The reagents used for comparison were produced by European and United States manufacturers in both the public and private sector and are widely used in routine ABO typing. The Scottish monoclonal reagents have been used successfully to ABO type over 500,000 blood samples in 7 centres within the UK, with no discrepant results.

ABO Blood-Group System↗

PubMed1988

Production and use of human monoclonal anti-D antibodies.

Rhesus haemolytic disease of the newborn is a condition which can result in intrauterine or perinatal death. Although the passive administration of therapeutic anti-D post-partum is a most effective method for the prevention of this condition, there is currently a shortage of immune plasma for the preparation of the therapeutic anti-D immunoglobulin product. In addition the availability of anti-D for use in blood grouping has also been reduced. The advances made in recent years in the techniques for the production of human monoclonal antibodies raise the possibility that human monoclonal anti-D-based products may provide solutions to both of these problems. There are now a number of reports of the production of stable cell lines secreting high titre human anti-D. In this review we consider the various strategies used in the production of human monoclonal anti-D-secreting cell lines, the basic properties of these reagents and their potential usefulness in blood grouping, in therapy and as research tools.

Antibodies, Monoclonal↗

PubMed1987

The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival.

Allogeneic rat mixed-thymocyte 48 h culture-conditioned medium (MTM) was used successfully in place of feeder cells for hybridoma production with the NS-1 and NS-0 plasmacytoma lines. It permitted lower concentrations of fused cells to be seeded, and supported the transition from 96 to 24 well plates. MTM improved the performance of poor sera during cloning. It also assisted the survival of cells that were sensitive to thawing from liquid nitrogen storage, and cells that had inadvertently been allowed to overgrow. Two rats could produce the equivalent of 1500-5000 ml feeder cell suspension according to the dilution used; 150-500 mice would be required to produce such a quantity of cells. Thus use of MTM entailed a considerable saving in mice and provided a secure supply of 'reagent', since a batch could be prepared, checked for sterility, frozen and stored indefinitely.

Animals↗

PubMed1986

The production and characterisation of a panel of ten murine monoclonal antibodies to human procoagulant factor VIII.

A panel of 10 murine monoclonal antibodies to procoagulant FVIII has been developed from the fusion of a single spleen. Balb/c mice were injected with a purified preparation of FVIII: Ag, and antibody production in sera and hybrid culture supernatants was monitored using a specific radiometric screening assay. The antibodies all inhibit FVIII clotting activity in normal plasma, and when immobilised on agarose retain their ability to recognise and bind the FVIII procoagulant protein. Studies on protein A-purified immunoglobulins demonstrate a range of properties within the panel of antibodies with regard to species cross-reactivity, clotting inhibition and immunoadsorption. The panel of antibodies has been used to screen heat-treated FVIII concentrates for the occurrence of heat-induced neoantigens.

Animals↗

PubMed1986

Characterisation of mouse monoclonal antibodies produced by immunisation with a single serotype component of a polyvalent Pseudomonas aeruginosa vaccine.

Mouse monoclonal antibodies raised by immunisation with a protective antigen extract from Pseudomonas aeruginosa serotype 1 varied in immunoglobulin isotype, in passive protective properties against infection by homologous P. aeruginosa serotype 1, and in cross-reactions in ELISA against antigen preparations from 15 other P. aeruginosa serotypes. All monoclonal antibodies with specificity in ELISA for the immunising antigen gave some degree of protection to mice against lethal infection by the homologous P. aeruginosa serotype. The IgG antibodies were more protective than the IgM antibodies.

Animals↗

PubMed1985

Enhancement of factor VIII-von Willebrand factor ristocetin cofactor activity by monoclonal antibodies.

Five monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any cross-reaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.

Animals↗

PubMed1985

ADCC lysis of human erythrocytes sensitized with rhesus alloantibodies. IV. Characterization of anti-D sera which are inactive in ADCC.

Certain anti-D sera, selected on the basis of their agglutination characteristics in vitro, fail to induce lysis of Rh(D) positive red cells by lymphocyte mediated antibody dependent cell mediated cytotoxicity (ADCC). Further investigation revealed that the non-lytic anti-D blocked in an antigen specific manner the effect of other anti-D sera which were normally lytic in ADCC. Absorption selection studies and fractionation of a non-lytic anti-D serum showed that the blocking effect was associated with IgG anti-D. Antigen binding and lymphocyte Fc-receptor binding studies indicated that the non-lytic anti-D was bound to Rh(D) positive red cells and enabled them to be bound by lymphocytes, but failed to mediate ADCC.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1983

Rhesus immunization in male volunteers: changes in lymphocyte functions following secondary immunizations in anti-D responders and non-responders.

After secondary immunizations of rhesus(D)-negative male volunteers with Rh(D)-positive red cells, changes were found in the in vitro transformation and antibody-dependent cell-mediated cytotoxicity (ADCC) capacities of the volunteers' lymphocytes. Responders, who produced anti-D, showed marked depressions of ADCC which were not found in non-responders. Responders and non-responders in general showed similar changes in lymphocyte transformation. The relationships between altered lymphocyte functions following immunization, immunoregulatory activity and responsiveness to the Rh(D) antigen are discussed.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1982