Xylogenesis: the birth of a corpse.

Xylogenesis is a complex developmental process culminating in programmed cell death as a truly terminal differentiation event. In Arabidopsis, the availability of vascular-patterning mutants, and the identification of genes and their products that are involved in cell specification, secondary-wall deposition and lignification, are providing clues to the functions of some of the sequences in the large expressed sequence tag databases derived from the xylem-rich tissues of trees. An in vitro system, the Zinnia mesophyll cell system, provides an alternative system for those cell-biological experiments that are difficult to tackle in intact plants. In particular, a combination of molecular-genetic and cell-biological approaches has made possible the elucidation of some of the features of plant programmed cell death.

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization).

Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains.

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.

Fourier-Transform Raman and Fourier-Transform Infrared Spectroscopy (An Investigation of Five Higher Plant Cell Walls and Their Components).

Infrared and Raman spectra of sequentially extracted primary cell walls and their pectic polymers were obtained from five angiosperm plants. Fourier-transform Raman spectrometry was shown to be a powerful tool for the investigation of primary cell-wall architecture at a molecular level, providing complementary information to that obtained by Fourier-transform infrared microspectroscopy. The use of an extraction procedure using imidazole instead of cyclohexane trans-1,2-N,N,N[prime],N[prime]-diaminotetraacetate allows the extension of the infrared spectral window for data interpretation from 1300 to 800 cm-1, to 2000 to 800 cm-1, and allows us to obtain Raman spectra from extracted cell-wall material. Wall constituents such as pectins, proteins, aromatic phenolics, cellulose, and hemicellulose have characteristic spectral features that can be used to identify and/or fingerprint these polymers without, in most cases, the need for any physical separation. The Gramineae (rice [Oryza sativa], polypogon [Polypogon fugax steud], and sweet corn [Zea mays]) are spectroscopically very different from the nongraminaceous monocotyledon (onion [Allium cepa]) and the dicotyledon (carrot [Daucus carota]); this reflects differences in chemical composition and cross-linking of the walls. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed.

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.

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.

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.

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.

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.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

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.

Fourier transform infrared microspectroscopy is a new way to look at plant cell walls.

Highly reproducible Fourier transform infrared (FTIR) spectra from both single onion (Allium cepa) cell walls and their constituent polymers were obtained under a variety of sampling conditions. The specificity of the chemical extraction sequence used in the preparation of the material was confirmed: pectins only are extracted by cyclohexanediaminetetraacetic acid and sodium carbonate, whereas xyloglucans are extracted by increasing concentrations of potassium hydroxide. There was very little contamination of the first potassium hydroxide extract with residual pectin. The low abundance of both phenolics and protein was also confirmed. The first sodium carbonate extraction almost completely removes esters remaining in the cell wall. We have demonstrated that FTIR spectroscopy can detect large conformational changes in pectic polymers on removal from the cell wall and on drying. FTIR spectroscopy provides a powerful and rapid assay for wall components and putative cross-links by identifying polymers and functional groups nondestructively in muro. The availability of micro-sampling and data acquisition techniques that permit subtraction of the blanket absorption of water make FTIR spectroscopy particularly suitable for studies of cell wall architecture. The use of polarizers with the microscope accessory permits determination of the orientation of particular functional groups with respect to the direction of cell elongation in carrot suspension cells.

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.

Developmental regulation of pectic epitopes during potato tuberisation.

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

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.

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.

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.