A Study of Protein and Glycoprotein Syntheses in Pre-implantation Mouse Embryos Using Mini-2D-Electrophoresis, Video Densitometer Scanning and Computer-Image Analysis

In the present study, proteins and glycoproteins of mouse embryos at 2-cell,morula and blastocyst stages were analyzed.The techniques of ~(35)S-Met incorporation,ConA antiserum-precipitating ConA-binding proteins, mini-2D-electrophoresis, fluorography,video densitometer scanning and the computer-lmage system were used for analyses.Results of the investigations indicated that proteins and glycoproteins were synthesized by the embryos in a gradual increase manner from 2-cell to blastocyst. A relatively large amount of glycoproteins was synthesized during the morula and blastocyst stages.Two specific small glycoproteins respectively with molecular weights about 6500 and 9000 and PIs at 5.0 and 6.5 were apparently synthesized in the blastocyst but not in the 2-cell or the morula.     

A possible modulation of acetylcholine receptors of embryonic chick muscle cells by alpha-bungarotoxin

Acetylcholine receptors were assayed with alpha-bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D-tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 x 10(-9)M and 2.7 x 10(-7)M at 25 degrees C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high-affinity receptors is about 200/micrometers2. A time course of toxin binding to receptors at 37 degrees C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was inactivating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.     

Cell surface changes accompanying myoblast differentiation

Myoblasts are mononucleated cells and associated with differentiation undergo cell fusion and become multinucleated. The current studies have examined cell surface dynamic changes of Concanavalin A lectin receptor mobility and the role of hormones in modulating myoblast differentiation. A uniform distribution of Con-A receptors is observed in undifferentiated cells when reacted with Con-A at 37 degrees C. Cells from differentiating cultures or fully differentiated myotubes reacted similarly at 37 degrees C show a significant redistribution of Con-A into patches, "caps," and endocytic vesicles containing Con-A. If undifferentiated and differentiated cells are first prefixed with glutaraldehyde then reacted with Con-A continuous distribution of Con-A is seen across the cell surface. This suggests redistribution of Con-A and its receptors occurs in differentiated cells reacted with lectin at 37 degrees C. It is further shown that insulin (10 microgram/ml) significantly enhances myoblast differentiation but that this occurs after an apparent stimulation of proliferation. In contrast to insulin, dexamethasone (10 micron and 100 micron) profoundly inhibits myoblast differentiation while having different effects on proliferation; 10 micron dex stimulates cell growth while 100 micron dex suppresses cell proliferation. Lastly, an extracellular filamentous matrix which binds Con-A is observed at the ultrastructural level in high density cultures. No significant redistribution of Con-A is observed on this matrix in distinction to the redistribution observed on the cell membrane in differentiated cells.     

Determination of the number of cells in preimplantation embryos by using noninvasive optical quadrature microscopy in conjunction with differential interference contrast microscopy.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.     

Counterflow affinity isotachophoresis on cellulose acetate membranes.

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.     

883 — Electrofusion of rat and mouse blastomeres

The limitations of the parameters of electrofusion of blastomeres in order to obtain viable embryos of rats and mice were ascertained for the usual single-pulse technique. Viability was observed upon applying pulse heights from 500 to 1500 V/cm and pulses widths of ≤ 0.3 ms. Afterwards renewed division of electrofused blastomeres started again within incubation of 24 h.Electrofusion of blastomeres was detected by treatment with an alternating current in the frequency range of 100–2000 kHz, 300 V/cm for ≤ 6 s. Increasing the voltage to above 300 V/cm or the time to above 6 s caused protoplasma denaturation, and finally disruption and even melting of the zona pellucida were observed. These experiments prove the possibility of electrofusion also through the zona pellucida without direct contact between the surrounding electrolyte medium and the inner membrane itself. Both techniques open up new bioelectrochemical possibilities of gene manipulations.     

Affinity chromatography with monolithic capillary columns. II. Polymethacrylate monoliths with immobilized lectins for the separation of glycoconjugates by nano-liquid affinity chromatography

Monolithic capillary columns with surface bound lectin affinity ligands were introduced for performing lectin affinity chromatography (LAC) by nano-liquid chromatography (nano-LC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylateco-ethylene dimethacrylate) and poly(glycidyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. Two lectins including concanavalin (Con A) and wheat germ agglutinin (WGA) were immobilized onto the monolithic capillary columns. The neutral monoliths with immobilized lectins exhibited lower permeability under pressure driven flow than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized lectins exhibited strong affinity toward particular glycoproteins and their oligosaccharide chains (i.e., glycans) having sugar sequences recognizable by the lectin. Due to the strong binding affinity, the monoliths with surface bound lectins allowed the injection of relatively large volume (i.e., several column volumes) of dilute samples of glycoproteins and glycans thus allowing the concentration of the glycoconjugates and their subsequent isolation and detection at low levels (approximately 10(-8) M). To further exploit the lectin monoliths in the isolation of glycoconjugates, two-dimensional separation schemes involving LAC in the first dimension and reversed-phase nano-LC in the second dimension were introduced. The various interrelated methods established in this investigation are expected to play a major role in advancing the sciences of "nano-glycomics".     

Interaction of lectins withYersinia pestis strains

The ability of lectins to interact with Yersinia pestis strains isolated from rodent fleas and human biological fluids, obtained from different geographic areas, was examined. Lectins of Canavalia ensiformis, Ulex europaeus, Phaseolus vulgaris, and Triticum vulgaris, as well as a new autochthonous lectin of Swartzia pickellii of undefined specificity, were used. Most of the Y. pestis strains did not agglutinate with U. europaeus or C. ensiformis lectin. However, P. vulgaris lectin agglutinated suspensions of all the bacillus strains used. Fifteen of the 19 strains tested positive for assays using S. pickellii lectin. It is believed this is the first report of Y. pestis strain agglutination by lectins. A similar agglutination pattern was obtained for lectins with specificity for oligosaccharides containing N-acetylglucosamine and S. pickellii lectin, which did bind to the affinity matrix chitin, a polysaccharide of N-acetylglucosamine. The use of bacterial strains and commercial lectins of defined specificity may be an approach to providing evidence about the lectin binding sites of undefined monosaccharide specificity.     

Separation of mistletoe lectins based on the degree of glycosylation using boronate affinity chromatography

A mixture of two mistletoe lectins (MLs) has been separated according to the degree of glycosylation using boronate affinity chromatography. The mistletoe lectins, mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) with degrees of glycosylation of 6.1 and 3.8%, respectively, were used in the investigation. MLI exhibited a higher retention time than MLIII due to its higher degree of glycosylation. Separation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The developed method may lead to new applications for the boronate affinity technique, as well as provide an alternative separation method for MLs.     

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope,LacdiNAc by Immune-Related Lectins

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.     

Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry

Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N?=?114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.     

Studies of fish zona pellucida by high-performance ion-exchange chromatography on agarose columns and free zone electrophoresis

By either free zone electrophoresis or high-performance ion-exchange chromatography on DEAE agarose, zona pellucida from Baltic small herring (Clupea harengus L.) was separated into several fractions. These fractions had very similar protein compositions, since on polyacrylamide gel electrophoresis in sodium dodecyl sulphate they all gave the same pattern: chiefly one major and three minor bands corresponding to proteins with the following estimated molecular weights: 78 000, 96 000 (the major component), 115 000, and 125 000. It is likely that these proteins constitute the so-called supramolecular complexes of zona pellucida from Baltic small herring. Only one electrophoretic and one chromatographic fraction gave precipitin arcs on immunodiffusion with rabbit antiserum against zona pellucida from the fish Aristichthys nobilis (Richardson).     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO(2) wafers was achieved under pH 11.6, 50mM CaCl(2), and at 70 degrees C. XG-ALG films presented mean thickness of (16+/-2)nm and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAlt adsorbed irreversibly onto XG-ALG forming homogenous monolayers approximately (4+/-1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue.     

Electrophoretic study of conformational changes of a human soluble beta-D-galactoside-binding lectin upon storage.

Human brain lectin (HBL), a beta-galactoside specific soluble lectin, was purified by affinity chromatography. An alkylated derivative of this lectin was also prepared. Both native and modified molecules were conserved at -20 degrees C in the presence or absence of beta-mercaptoethanol, a reducing agent which was described to maintain the lectin activity in vitro or in the presence of beta-mercaptoethanol and lactose. The impact of storage conditions, over one year, on the native and derivated lectins, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and titration curve, using the PhastSystem (Pharmacia). Western-blot analysis using an anti-HBL antibody and size-exclusion high performance liquid chromatography were used to complete the study. The subunit M(r)s were estimated before freezing (T0) and after three and twelve months (T3, T12). They were comparable for all preparations. In all samples tested, isoelectric focusing demonstrated the existence of at least three acidic proteins, with the pI ranging between 4.7-4.9. Titration curves clearly showed pH-dependent conformational changes, resulting in a panel of differently charged molecular species, some of which may be related to different oxidative states of the cysteine residues. We concluded that lectin can be stored at -20 degrees C for at least one year before use as a reagent since the modifications revealed by electrophoretic analysis do not alter the hemagglutination activity and carbohydrate binding properties. The immunoreactivity also remained unchanged.     

Detection of cytotoxic activity of lectin on human colon adenocarcinoma (Sw480) and epithelial cervical carcinoma (C33-A)

Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius) lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480); By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines.     

Profiling the Cell Surface Glycome of Five Fungi Using Lectin Microarray

Fungal surface glycans play roles in cell protection and interaction. Our knowledge of fungal glycans, however, is limited to model yeasts and a few medically/industrially important fungi. We evaluated the usefulness of a lectin microarray in analysis of live cell surface glycans and then applied the technique to glycome of varieties of yeast-form fungi from various phyla and subphyla. We found glycan profiles are different among and within taxonomic groups. For example, Saccharomycotina were classified into two groups, one bound to mannose-specific lectins and the other bound to galactose/N-acetylgalactosamine-specific lectins. In Basidiomycota, Pucciniomycotina yeasts were distinguished from other subphylum members in binding to fucose-specific lectins. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.     

LECTIN RECEPTORS ON ROD AND CONE MEMBRANES

Abstract— The oligosaccharides of rod and cone membranes were investigated with the aid of fluorescence and ¹²⁵I-labeled lectins. Additionally, the ability of lectins to cause agglutination in rod outer segment (ROS) suspensions was used as an index for the presence of the corresponding lectin receptors. The specificities of lectin-ligand interactions were determined from studies of inhibition by various haptene sugars. The membranes of both rods and cones have receptors for Con A, PNA, RCA-120, RCA-60, SBA and WGA. The affinity of PNA for accessory cones is much higher than for the principal cones. There do not appear to be receptors for UeA and LTA on rods or cones. Additionally, receptors for HPA and DBA were identified on ROS. These results suggest the existence of the following sugar residues:

The binding of Con A and WGA to ROS membrane proteins electrophoresed on SDS-polyacrylamide gels was also investigated. In addition to rhodopsin, these lectins also bind to the 291000-dalton protein, indicating that it is a glycoprotein containing mannose and GlcNAc.     

Defective histone H3K27 trimethylation modification in embryos derived from heated mouse sperm

The mouse sperm genome is resistant to in vitro heat treatment, and embryos derived from heated sperm can support full-term embryonic development, but the blastocyst rate and implantation rate are lower compared to embryos derived from fresh sperm. In the present study, the patterns of DNA methylation, histone H4K12 (ACH4K12) acetylation, H3K9 trimethylation (H3K9-TriM), and H3K27 trimethylation (H3K27-TriM) in preimplantation embryos derived from 65 °C-heated sperm were investigated. Although no evident changes in global DNA methylation, histone H4K12 (ACH4K12) acetylation, and H3K9 trimethylation (H3K9-TriM) were found, significantly lower levels of H3K27-TriM, which was thought to be one of the reasons for low efficiency of mouse cloning, were found in the inner cell mass of heated-sperm derived blastocysts. Thus, defective modification of H3K27-TriM might contribute to compromised development of embryos derived from heated sperm.     

A microfluidic method for removal of the zona pellucida from mammalian embryos

Many in vitro procedures involve manipulation of the zona pellucida (chimerics, transgenics, biopsy). We have demonstrated a microfluidic channel network to precisely control (spatially and temporally) the delivery of chemical treatments for the removal of the zona pellucida. Building devices in polydimethylsiloxane (PDMS) with channel dimensions on the same order as that of the embryo diameter (approximately 120 microm) allows precise control of the local fluid environment. The system uses pressure driven flows to control embryo positioning, embryo movement, and plug formation. Zona removal is achieved by briefly washing a plug of lysing agent (acid Tyrode's medium) over the embryo.