Binding sites of Ulex europaeus-lectin I in human parotid gland

Summary Twenty non-neoplastic parotid glands (removed during neck dissection for regional tumours) were examined for cellular and subcellular binding sites of Ulex europaeus-lectin I (UEA-I), a lectin reported to be specific for -L-fucose. For light microscopy, an extended peroxidase-antiperoxidase method was applied; for the evaluation of the subcellular localization of bound lectin, three of these glands were examined following immunocryoultramicrotomy and staining by the protein A-gold technique.In addition to the known cytoplasmic affinity of UEA-I for capillary endothelium, acinar cells bound the lectin within the cytoplasmic compartment; the number and distribution of stained acinar cells varied among individuals. Furthermore, cytomembrane-bound labelling that occurred most markedly at the luminar surface was observed in striated-duct epithelium.Using the electron microscope, protein A-gold particles were seen in zymogen granules and in Golgi cisternae of serous acinar cells; primary saliva secreted in the lumina exhibited strong labelling; serous acinar cells had binding sites on their cell membranes, striated-duct epithelium had binding sites on its surface membrane and in the vicinity of apical vesicles. Our results show that UEA-I is a useful tool for the study of the structure and functional states of the parotid gland epithelium and its associated pathological alterations.Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 65th birthday     

Formation of the nuclear envelope during mitotic anaphase inSpirogyra

The formation of the nuclear envelope in the mitosis ofSpirogyra was studied with an electron microscope. The nuclear envelope was disrupted around the spindle equator in the metaphase. Many small vesicles were observed in the metaphase spindle. These vesicles surrounded the masses of chromosomes and nucleolar substance in the early anaphase, and they fused with each other to form daughter nuclear envelopes during the early anaphase. The formation of new envelopes from small vesicles at such an early mitotic anaphase is reported here for the first time. The possible origin of these vesicles is also discussed.     

Specific interaction of lectins with liposomes and monolayers bearing neoglycolipids

The interaction of three lectins (wheat germ, Ulex europaeus I, and Lotus tetragonolobus agglutinins: WGA, UEA-I and LTA) with either N-acetyl-D-glucosamine or L-fucose neoglycolipids incorporated into phospholipid monolayers and liposome bilayers was studied at the air/water interface and in bulk solution.The results show that for both systems studied, synthesized neoglycolipids were capable of binding their specific lectin and that, in general, the binding of lectins increased with the increase in the molar fraction of the saccharide derivative incorporated in either the monolayers or bilayers. However, whereas for UEA-I, molecular recognition was enhanced by a strong hydrophobic interaction, for WGA and LTA successful recognition was predominantly related to the distance between neighboring sugar groups. The observed lengthy adsorption times of these lectins onto their specific ligands were attributed to interfacial conformational changes occurring in the proteins upon their adsorption at the interfaces.     

The macronuclear envelope of Tetrahymena pyriformis GL in different physiological states

Summary Macronuclear envelopes were isolated from the ciliated protozoan Tetrahymena pyriformis GL, negatively stained and examined in the electron microscope. The frequency of central granules in the macronuclear pores was evaluated in five different physiological states: (1) stationary phase of growth, (2) exponential phase of growth, (3) heat-synchronized cultures at the end of the heat-synchronization treatment, (4) heat-synchronized cultures at the beginning of the first division, (5) heat-synchronized cultures at the end of the first division.The percentage of pores containing a central granule was markedly enhanced in heatsynchronized cultures at the end of the first division, i.e. a state known for an increase in ribosome formation. Actinomycin D was found to cause a significant decrease in central granule frequency.The observed alterations in central granule frequency seem to confirm the hypotheses which consider the central granule as representing a ribonucleoprotein particle in transit from nucleus to cytoplasm through the nuclear pore.For careful technical assistance I am indebted to Miss Marianne Whiter as well as to Drs. H. Falk, W.W. Franke and P. Sitte for helpful discussions. This work was supported in part by the Deutsche Forschungsgemeinschaft.     

Chloroplast envelopes as affected by light or dark pretreatment of pea plants

Glutaraldehyde fixation in 0.33 M sorbitol without any buffer reveals changes in the staining properties of the envelopes of chloroplasts of pea plants kept in the light or in the dark prior to fixation. After dark pretreatment the outer double membrane of the chloroplast does not adsorb heavy metals, resulting in a white unstained rim instead of the usual membrane. All other membranes of the cell, including chloroplast grana, are not affected and stain normally. Light pretreatment of the plants allows the usual staining of the outer membrane of the chloroplats. Fixation carried out in the medium usually used to isolate intact CO₂ fixing chloroplasts (sorbitol+buffer+ions) reverses the above process and results in unstained envelopes of chloroplasts from preilluminated leaves, while the envelopes of chloroplasts from leaves kept in the dark stain normally. Glutaraldehyde-fixed chloroplats isolated from preilluminated leaves show a very basic isoelectric point during electrofocusing, while fixed chloroplasts from predarkened tissue exhibit an isoelectric point at about pH 7.     

Reversible,lectin-mediated immobilization of plant protoplasts on agarose beads

A technique is described for the reversible, lectin-mediated immobilization of plant protoplasts on agarose beads. Cyanogen-bromide-activated agarose beads were coated with protein (gelatine or bovine serum albumin) and lectins were subsequently linked to the protein layer using glutaraldehyde. The technique has possible applications in protoplast fusion-product isolation, cellrecognition studies, and membrane isolation.Abbreviations BSA bovine serum albumin - Con A concanavalin A - FDA fluorescein diacetate - PNA peanut agglutinin - WGA wheat-germ agglutinin     

Optimization of use of UEA-1 magnetic beads for endothelial cell isolation

Most endothelial cells (EC) in the body belong to the microvasculature. Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specificity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericytes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1), or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, operative conditions remain to be optimized to obtain pure cultures of EC.We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences are studied of time, temperature, cell concentration, and number of beads per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts (HSF), either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing proliferation after isolation. Results, expressed as isolation efficiency (from 40% to 90%) allowed us to select a 15-min incubation time at 4°C with rotary agitation, an optimal concentration of 4 x 10⁵ cells/ml, and an optimal cell:bead ration of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportions of 2.5:97.5 allowed us to obtain a pure HUVEC population in subsequent culture.Abbreviations UEA-1 Ulex europaeus agglutinin-1 - EC endothelial cells - HUVEC human umbilical vein endothelial cells - HSF human skin fibroblasts - MPC magnetic particle concentrator - IE isolation efficiency     

Transmural exocytosis in maize root cap

Summary The maize root cap is a tissue known for its high production of a fucose-rich slime. At the cellular periphery, two kinds of components exist which are indistinguishable: the cell wall barrier and the slime which passes through. Two complementary probes were used, both at the light and the electron microscope level, in order to distinguish the different components. The lectinUlex europaeus agglutinin I was used as a probe targetting the slime and the enzyme cellulose 1,4--D-glucan cellobiohydrolase I was used to probe the cellulose framework. Both probes were used either alone or sequentially for double labeling. The cytochemical PATAg test was optionally used with the enzyme-gold complex labeling. After several technical improvements (multistep method, increase in accessibility), UeA I was used to follow the exocytic pathway of the slime from the Golgi apparatus to the exterior of the cell. The results indicate the occurrence of at least two populations of Golgi apparatus vesicles, and one is directly engaged in the transport of the fucoserich slime. The slime accumulated in pockets between the plasmamembrane and the outer tangential cell wall. The CBH I-gold complex showed the existence and the maintenance of a thin but continuous cellulosic layer, even when the cells slough. The double labeling showed the fucose-rich compounds within the cell wall. Data emphasize the role of the cell wall as a filtering barrier and a mechanical protection in the course of differentiation.Abbreviations CBH I EC 3.2.1.91, cellulose 1,4--D-glucan cellobiohydrolase I - CMC carboxymethyl cellulose - CPB citrate phosphate buffer - FITC fluorescein isothiocyanate - IC internal cell - PA-TAg periodic acid-thiocarbohydrazide-silver proteinate - PBS phosphate buffered saline - PC cell with accumulation pockets - PEG polyethyleneglycol - SC sloughed cell - UeA I Ulex europaeus agglutinin I - VI UeA I-labelled Golgi-derived vesicles - V2 UeA I-unlabelled Golgi-derived vesicles     

The recognition of three different epitopes for the H-type 2 human blood group determinant by lections ofUlex europaeus,Galactia tenuiflora andPsophocarpus tetragonolobus (Winged Bean)

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc(1–2)Gal(1–4)GlcNAcMe) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc(1–2)Gal(1–4)XylMe was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.Abbreviations Me methy - Bn benzyl - Ac acetyl - Bz benzoyl - n-Bu n-butyl - NMR nuclear magnetic resonance - the GlcNAc Gal and Fuc residues of the H-type 2 trisaccharide are designated as the a, b and c structural units, respectively. This is paper XV in a series devoted to molecular recognition.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.This work was supported under the LINK programme in Selective Drug Delivery and Targeting, funded by SERC/MRC/DTI and industry (SERC grant GR/F 09747). M.A.J. was also supported by a Wellcome Postdoctoral Research Followship (039684/Z/93/Z). Additional support was provided by the Royal Society for equipment.     

ATP-induced budding of nuclear envelope in vitro

Summary Fractions enriched in intact nuclei and nuclear fragments isolated from etiolated hypocotyls of soybean responded in vitro to ATP plus a concentrated fraction of cytoplasmic proteins by formation of ca. 50–70 nm buds and vesicles resembling those observed to bud from the outer membrane of the nuclear envelope in situ at regions of nuclear envelope-Golgi apparatus interface. Similar vesicles are normally considered to function in the transfer of materials from the outer membrane of the nuclear envelope to cis elements of the Golgi apparatus.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

Observations on North-West European limestone grassland communities: Phytosociological and ecological notes on chalk grasslands of Southern England

Summary In 1970 chalk grasslands were studied in southern England, according to the principles of the French-Swiss School. The communities described belong to the associationCirsio-Brometum Shimwell 1971, emend., allianceMesobromion erecti (Br. Bl. & Moor 1938) Oberd. 1957, orderBrometalia W. Koch (1926 n.n.) Br.-Bl. 1936, of the classFestuco-Brometea Br.-Bl. 1936, emend. Tüx. 1961. Within theCirsio-Brometum three new sub-associations are distinguished: 1) subass. nov.anthoxanthetosum odorati; 2) subass. nov.Cornetosum sanguineae; and 3) subass. nov.campanuletosum rotundifoliae.Special attention is paid to the relationship that exists between the management of these communities and the seral stages that lead to the development of serub following the decreasing influence of grazing. Ulex europaeus scrub occurring on calcareous soil was also studied. The cryptogams of the chalk grasslands, mainly Musci, were investigated in detail.Plant nomenclature follows Heukels & van Ooststroom (1970) or for taxa not mentioned by these, Clapham et al. (1968) for phanerograms and Margadant (1959) and van der Wijk et al. (1969) for bryophytes.Contribution to the Symposium on Plant species and Plant communities, held at Nijmegen, 11 12 November 1976, on the occasion of the 60th birthday of Professor Victor Westhoff.     

Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum

Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations endoH endoglycosidase H - ER endoplasmic reticulum - Mr relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TBST Tris-buffered saline containing Tween 20 We thank Debra Donaldson for her contribution to the PHA gene constructions. This work has been supported by grants from the National Science Foundation (Cell Biology) and the Department of Energy (DE-FG03-86ER13497) to Maarten J. Chrispeels. The assistance of the staff of the Electron Microscope Laboratory, USDA, Beltsville is gratefully acknowledged.     

Host specificity, establishment and dispersal of the gorse thrips, Sericothrips staphylinus Haliday (Thysanoptera: Thripidae), a biological control agent for gorse, Ulex europaeus L. (Fabaceae), in Australia

Sericothrips staphylinus was released as a biological control agent for Ulex europaeus in New Zealand and Hawaii following tests on ca. 80 plant species which showed it was narrowly oligophagous. To determine the suitability of S. staphylinus for release in Australia, further host specificity tests were conducted on 38 species and cultivars of Australian plants. These tests confirmed that S. staphylinus would feed only on U. europaeus in Australia and, following formal approval, was released in Tasmania during January 2001. To develop an optimal release strategy for S. staphylinus under Australian conditions, a field trial based on an earlier New Zealand study was conducted by replicating releases of 10, 30, 90, 270 and 810 adults. Results showed that population growth, reproduction rate and the number of S. staphylinus recovered 14 months after release can be non-linear functions of release size and establishment could be achieved with as few as 10 thrips. As S. staphylinus is easily cultured ca. 250 thrips were chosen as the minimum number for release because, based on a negative binomial model, this release size produced close to the maximum population growth. Surveys in early 2007 recovered S. staphylinus from 80% of 30 sites in Tasmania, the post release period ranging from 1 to 6 years. However, densities were low (<1 thrips/cm of tip growth) with no evidence of visible plant damage. The maximum dispersal range was 180–250 m after 38 months. At all the other sites, dispersal was estimated at less than 120 m. It is possible that S. staphylinus populations are still in the lag phase of their establishment before starting to increase rapidly and disperse. However, the survey results support a recent Tasmanian study which indicated that S. staphylinus is a sedentary, latent species characterised by steady densities and low levels of damage to its host plant. Its efficacy as a biological control agent on gorse may be restricted primarily by ‘bottom up’ effects of plant quality limiting its rate of natural increase and an inability of the thrips to reach large, damaging populations under field conditions.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

The development of nuclear vacuoles during meiosis in plants

Vacuoles formed by the invagination of the inner membrane of the nuclear envelope have been observed during meiotic prophase in a wide range of plants. In the angiosperm Lycopersicon their formation was found to coincide with the completion of synaptonemal complex formation, and this timing is analogous to that observed during this stage in the silkworm Bombyx. The implications of this activity in relation to the process of chromosome movement are discussed. In the gymnosperm Pinus, the heterosporous fern Marsilea and homosporous ferns Pteridium and Dryopteris the formation of nuclear vacuoles begins much earlier, coinciding with the condensation of chromatin during leptotene. They enlarge and become more elaborate as meiosis proceeds, and may eventually become detached from the nuclear envelope. It is therefore thought unlikely that theyfulfil functions connected with chromosome movement in the manner proposed for the silkworm and the tomato. During diplotene/diakinesis they contain electron-opaque granules and fibrils, and the possible origin and significance of this material is discussed.     

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Perinuclear and nuclear envelope localizations of <Emphasis Type="Italic">Arabidopsis</Emphasis> Ran proteins

Using phragmoplastin-interacting protein 1 (PhrIP1) as bait, we isolated an Arabidopsis cDNA encoding Ran2, a small Ras-like GTP-binding protein. The interaction between PhrIP1 and Ran2 was confirmed by an in vitro protein–protein interaction assay with purified Ran2 and PhrIP1. The plant Ran2 shares high sequence homology, 78 and 86% at the amino acid level, with human Ran/TC4 and C. elegans Ran, respectively. Our results obtained from enzyme assays and Western blot analysis show that Ran2 has intrinsic GTPase activity and is present in the soluble fraction of Arabidopsis seedling extract. Fluorescent microscopy using anti-Ran2 antibody revealed that the Ran protein is localized in the perinuclear region with the highest concentration at the nuclear envelope. In contrast to its animal counterparts that are present in the nucleoplasm, the Ran protein is absent inside the nucleus. These results suggest that plant Ran proteins may be involved in mediation of nucleocytoplasmic transport and assembly of the nuclear envelope after karyokinesis in plant cells.