Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.     

Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le(x); Gal beta 4[Fuc alpha 3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by approximately 70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le(a); Gal beta 3[Fuc alpha 4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le(x)- and Le(a)-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le(x)-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le(a)-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le(x)-containing neoglycoproteins. In contrast, neoglycoproteins containing beta4-lactosamine (Gal beta 4GlcNAc), Lewis B (Fuc alpha 2Gal beta 3[Fuc alpha 4]GlcNAc), and sialyl-Le(x) glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le(x)- and Le(a)-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le(x)- and Le(a)-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G(i) protein. Thus, Le(x)-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le(a)-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.     

Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines

Summary— In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either α-l -fucosyl or α-l -rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to α-l -rhamnosyl-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4°C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, α-l -Rha-BSA and F-, α-l -Fuc-BSA were efficiently internalized. Thereafter, α-l -rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL₁α, IL₁β, IL₂ and TNF) which are known as biological response modifiers of keratinocytes.     

Tissue distribution of radioiodinated neoglycoproteins and mammalian lectins

Quantitation of tissue distribution of radioiodinated neoglycoproteins 1 h after intravenous injection into mice allowed to evaluate their suitability to uncover potential selectivity in tracer retention. Variations within the panel of neoglycoproteins were introduced to the carbohydrate determinant, its density and linkage to the carrier. Five arrays of neoglycoproteins, encompassing up to twelve different carbohydrate moieties were used. The individual response on the level of organ content showed differences, accounted for by carbohydrate structure and density. However, increase in sugar density eventually caused general decrease in tissue retention, emphasizing the importance of synthetic parameters. Attachment of sugar residues to the spacer via primarily the C-6 group of monosaccharides led to rather prolonged survival in circulation of the resulting neoglycoprotein compared to the application of neoglycoproteins with p-aminophenyl glycosides as derivatives for coupling. Besides applying neoglycoproteins tissue uptake was also measured for several organs, when four mammalian lectins were employed as radiotracers. These lectins bind to cellular carbohydrate ligands, namely beta-galactosides, alpha-fucosides or heparin. Differences were measured for retention in liver, kidneys, spleen, stomach, thymus and bone marrow. The distinct properties of different tissues with respect to binding of neoglycoproteins as well as to endogenous lectins, exhibiting a certain degree of selectivity, are a step within the framework to attempt to therapeutically exploit the carrier potential of probes by recognitive protein-carbohydrate interactions.     

Phosphorylation associated with succinate decarboxylation to propionate in Ascaris mitochondria

Mono-, bis-, and tris-β-d-galactopyranosides of (2-(5-hydrazinocarbonylpentanamido)-2-(hydroxymethyl)-1, 3-propanediol were synthesized. Treatment of the glycosides with nitrous acid gave the corresponding acyl azides which were coupled to bovine serum albumin to form neoglycoproteins. These derivatives were tested for their inhibitory action on (i) the d-galactose binding by rabbit liver membrane, (ii) the corresponding binding by the isolated binding protein, and (iii) the corresponding uptake by intact rat hepatocytes. In these systems, the neoglycoprotein with attached tris-galacto was a better inhibitor than the bis derivative, which in turn was more effective than the mono derivative per each ligand. However, the order is reversed when the inhibitory action is expressed on the basis of d-galactosyl residue.     

Fluorescent derivatives of ganglioside GM1 function as receptors for cholera toxin

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.     

A critical evaluation of neoglycoprotein binding sites in vivo and in sections of mouse tissues.

Endogenous lectins are reported to play a vital role in cell to cell communication. Their distribution in tissues has been widely studied by the use of labelled neoglycoproteins. In the present study, labelled neoglycoproteins were used on fixed and unfixed tissue sections and the results were compared with those observed after i.v. application of neoglycoproteins in mice. The study indicates that neoglycoprotein binding to tissue sections is not inhibited by application of the simple monosaccharides that were used to synthesize them. Furthermore the binding of neoglycoproteins following i.v. application into mice is rather limited. It is concluded that neoglycoproteins, which are synthesized using simple monosaccharides, do not provide a sensible tool to detect endogenous lectins in animal tissue sections. This is in sharp contrast to the results of most other studies reported in the literature.     

A critical evaluation of neoglycoprotein binding sites in vivo and in sections of mouse tissues

Summary Endogenous lectins are reported to play a vital role in cell to cell communication. Their distribution in tissues has been widely studied by the use of labelled neoglycoproteins. In the present study, labelled neoglycoproteins were used on fixed and unfixed tissue sections and the results were compared with those observed after i.v. application of neoglycoproteins in mice. The study indicates that neoglycoprotein binding to tissue sections is not inhibited by application of the simple monosaccharides that were used to synthesize them. Furthermore the binding of neoglycoproteins following i.v. application into mice is rather limited. It is concluded that neoglycoproteins, which are synthesized using simple monosaccharides, do not provide a sensible tool to detect endogeneous lectins in animal tissue sections. This is in sharp contrast to the results of most other studies reported in the literature.     

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.     

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.     

Sugar receptor mediated drug delivery to macrophages in the therapy of experimental visceral leishmaniasis

Methotrexate (MTX) conjugate of a neoglycoprotein, mannosyl bovine serum albumin, containing an average of 30 moles of MTX per mole of neoglycoprotein was taken up efficiently by murine peritoneal macrophages through cell surface mannosyl receptors. The conjugate strongly inhibits the growth of Leishmania donovani inside macrophages, with 50% inhibitory dose of 0.11 micrograms/ml MTX, which makes it 100 times more active than free MTX (50% inhibitory dose of 12.1 micrograms/ml). MTX conjugated to BSA or other non-specific neoglycoproteins like galactose-BSA and glucose-BSA have leishmanicidal effects comparable to free MTX. Moreover, in a murine model of experimental visceral leishmaniasis, the drug conjugate reduced the spleen parasite burden by more than 85% in a 30 day model whereas the same concentration of free drug caused little effect. The results demonstrate that neoglycoproteins may be useful as carriers for receptor mediated drug delivery to treat macrophage associated diseases.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties

We address the question whether the two natural types of branching in complex-type triantennary N-glycans differ in ligand properties. Toward this end, we prepared the set of pergalactosylated undecasaccharides and derivatives with alpha2,3/6-sialylation by chemoenzymatic synthesis. Conjugation resulted in neoglycoproteins which were tested in assays with lectins/antibodies, cultured cells and animals. Solid-phase assays with galactoside-specific proteins (a plant toxin, galectins and an antibody fraction) disclosed that the branching mode did not significantly affect affinity. However, compared to previous studies under identical conditions increase in antennae number and presence of substitutions in biantennary N-glycans altered KD-values with differences between receptors. Neoglycoprotein binding to cells of eight human tumor lines was sensitive to N-glycan branching. Staining intensity revealed pronounced branch-mode-dependent differences in four cases. Biodistribution profiles in mice uncovered dramatic changes in clearance rates with prolonged serum presence associated with type II branching of sialylated N-glycans and markedly increased uptake of neoglycoproteins with type I-branched N-glycans into liver, spleen, heart and lungs. This part of the study is relevant for rational glycoengineering of pharmaproteins. In general, our study supports the concept to view details of N-glycan structure, here branching, as a means to modulate ligand properties.     

Ultraviolet Rays Induced Expression of Lectins on the Surface of a Squamous Carcinoma Keratinocyte Cell Line

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either α-l-rhamnosyl or α-d-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sun-like doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. α-l-Rhamnose-specific receptor on SCL-1 could not be distinguished from α-d-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required ade novoprotein synthesis process.     

Correlations between the binding of neoglycoproteins,bovine serum albumin (BSA) and lectins in 10 to 13-day-old mouse embryos

Summary In the present work we compared the appearance of carbohydrate binding sites for mannose, maltose, sialic acid and N-acetyl-glucosamine in the 11 to 13-day-old mouse embryo with the appearance of BSA and lectin binding sites. The carbohydrate-binding sites were localized with FITC-coupled neoglycoproteins, synthesized by chemical glycosylation of bovine serum albumin (BSA). These localizations were compared with binding of the FITC-labelled unglycosylated BSA. Furthermore the localizations of neoglycoprotein and BSA binding sites were correlated with binding of the FITC-labelled lectins WGA, RCA I and Con A. Initial appearance of neoglycoprotein binding sites occurred in the lens capsule of the 13 day old mouse embryo. Binding sites for the unglycosylated BSA appeared earlier, i.e. already in the 12-day-old embryo, in the basement membranes of the choroid plexus and the lung bud and lectin binding sites were seen in these structures in the 11-day-old embryo. The staining of the basement membrane and the lens capsule for BSA binding sites in the 12-and 13-day-old embryos correspond to WGA binding to these membranes. From these results we concluded that 1) specific carbohydrates which are probably involved in embryonic development appear much earlier in the embryo than the endogenous lectins which are able to react with these carbohydrates and 2) BSA is a protein which like WGA probably binds N-acetylglucosamine or sialic acid moieties.     

Binding of the Bacillus sphaericus mosquito larvicidal toxin to cultured insect cells

Using both fluorescent labelled toxin and antibody--secondary antibody techniques, the Bacillus sphaericus toxin was found to bind strongly to susceptible Culex quinquefasciatus cells, but far less strongly to cells of insensitive insects. An insensitive clone of the C. quinquefasciatus cell line was discovered which bound toxin efficiently. The toxin was bound in the cold to sensitive cells and these cells could be rescued from cytotoxicity for ca. 15 min after warming, by which time toxin appeared to be internalized. Binding was saturable. This toxin is apparently internalized by receptor-mediated endocytosis, probably involving a glycoprotein receptor containing N-acetyl-D-glucosamine. Evidence for toxin binding to lipids was not found. Antibody appeared to detect internalized toxin, and high concentrations of sugars inhibited cytotoxicity; these results along with evidence from a recent ultrastructural study suggest that this toxin may form pores in the cell membrane.     

Selective macrophage activation by muramyldipeptide bound to monoclonal antibodies specific for mouse tumor cells

Summary IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP).This statement is based on experiments using two IgM monoclonal antibodies selected for their ability to bind L1210 leukemia cells (F₂-10-23-IgM) and 3LL Lewis lung carcinoma cells (6B6-IgM) specifically, as shown by flow cytofluorometry analysis.The MDP-IgM conjugates, containing 45 MDP molecules per IgM molecule, were prepared by allowing MDP-hydroxy-succinimide ester to react with IgM monoclonal antibodies.The MDP-IgM conjugates are shown to bind to relevant tumor cells and to induce the activation of thioglycolate-elicited peritoneal mouse macrophages leading to 80% growth inhibition of target cells at optimum concentrations of bound MDP. These concentrations of bound MDP were 10 times lower than the concentration of free MDP, giving a maximum activation that is limited to 20% growth inhibition.No macrophage activation was evidenced when tumor cells were incubated in the presence of irrelevant MDP-IgM conjugates and macrophages or when macrophages were preincubated in the presence of MDP-IgM conjugates and then incubated in the presence of relevant or irrelevant tumor cells but in the absence of the MDP-IgM conjugates.The reported results are discussed with reference to the mechanism of activation of macrophage by muramyldipeptide and to the usefulness of such MDP-IgM conjugates as potential antitumor agents in cancer therapy.Abbreviations ADMC antibody dependent macrophage mediated cytotoxicity - F-GAM fluoresceinylthiocarbamyl goat anti mouse antibody - -Man-BSA -mannopyranosyl-phenylthiocarbamyl bovine serum albumin containing some 25 mannose residues (neoglycoprotein) - MDP muramyldipeptide, 2-acetamido-3(2-0-d-lactyl-l-alanyl-d-glutamyl amine) glucopyranose - MDP-F₂-10-23-IgM Murine monoclonal antibody specific of L1210 leukemia cells and substituted with 45 MDP molecules - MDP—6B6-IgM Murine monoclonal antibody specific of 3LL Lewis lung carcinoma cells and substituted with 45 MDP molecules - MEM minimal essential medium - TDM Trehalose-dimycolate     

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.     

糖肽对小鼠Lewis肺癌实验性转移作用的初步病理观察

我们曾报道从小鼠Lewis肺癌组织通过蛋白水解酶及分子筛层析分离的总糖肽,在体外可明显地抑制某些肿瘤细胞及分离的层粘连蛋受体与基膜成分层粘连蛋白的识别和结合。本文报告将此糖肽与Lewis肺癌细胞混合,通过尾静脉注入小鼠体内,对实验性癌转移的抑制作用。初步病理结果表明,此糖肽几乎可以完全抑制实验性转移瘤的形成,保护小鼠不死于癌转移。提示糖肽可能具有阻断癌转移之作用。将实验组一部分存活小鼠再行同种癌细胞皮下接种,可以照常成瘤。表明糖肽阻抑实验性癌转移的效能可能并非调动了宿主的免疫机制所致。糖肽还可减慢皮下接种的癌细胞的生长速度,但对癌细胞并无直接毒性作用。