Influence of 3-hydroxymethyl xylitol, a novel antidiabetic compound isolated from Casearia esculenta (Roxb.) root, on glycoprotein components in streptozotocin-diabetic rats.

Casearia esculenta root (Roxb.) is widely used in traditional system of medicine to treat diabetes in India. An active compound, 3-hydroxymethyl xylitol (3-HMX), has been isolated, and its optimum dose has been determined in a short duration study and patented. In addition, the long-term effect of 3-HMX in type 2 diabetic rats on antihyperglycemic, antioxidants, antihyperlipidemic, and protein metabolism and kidney marker enzymes was investigated, and its effect was shown previously. In this study, we investigated the effect of 3-HMX on plasma and tissue glycoproteins in streptozotocin-diabetic rats. Animals were divided into five groups viz., control group, 3-HMX (40 mg/kg of body weight) treated group, diabetic group, diabetic+3-HMX (40 mg/kg of body weight), and diabetic+glibenclamide (600 µg/kg of body weight). 3-HMX was administered orally at a dose of 40 mg/kg of body weight for 45 days. The study shows significant increases in the level of sialic acid except kidney and elevated levels of hexose, hexosamine, and fucose in the liver and kidney of diabetic rats, and the treatment with 3-HMX and glibenclamide showed reversal of these parameters toward normalcy. Thus, the study indicates that 3-HMX possesses a significant beneficial effect on glycoprotein components.

Multi-endpoint biological monitoring of phosphine workers.

The pesticide phosphine (PH(3)) is a suspected carcinogen and a known clastogen which has been shown to produce chromosome damage in agricultural workers. To confirm and extend these results we evaluated 22 phosphine appliers and 26 controls matched for age and smoking status. Two independent methods were used to evaluate exposure: fluorescence in situ hybridization (FISH) with whole-chromosome paints of chromosomes 1, 2, and 4 labeled in a single color to quantify translocations in peripheral lymphocytes, and the glycophorin A (GPA) assay to quantify phenotypically mutant (NØ or NN) erythrocytes. No differences in the frequency of translocations were found in the phosphine appliers compared to the controls, and no effect of cigarette smoking was observed. However, a significant increase in the frequency of translocations with age (P<0.0001) was seen. No effect of phosphine exposure or cigarette smoking was observed in the GPA assay. These results are in contrast to previous findings from this same population which showed an increase in chromosome aberrations among phosphine appliers. The results are most easily interpreted as supporting the effectiveness of the personal protective equipment that is now worn by the workers but which was not employed prior to and during the earlier studies.

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Polyethylene glycol-coated red blood cells fail to bind glycophorin A-specific antibodies and are impervious to invasion by the Plasmodium falciparum malaria parasite.

This study was designed to assess the binding of glycophorin A-specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion by Plasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A-specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A-specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.

A new method to evaluate in vitro myelotoxicity of antitumour agents in the first steps of drug development.

Research focused on the development of new anticancer agents has been based mainly on the assessment of the antitumour activity. This yields a large number of newly developed drugs endowed with good antitumour properties, but heavy side-effects on myelopoiesis. In this work, we validate a new method potentially useful to assess myelotoxic effect of newly developed agents. The proposed technique uses peripheral blood CD34+ cells as source of haematopoietic progenitors. These cells are grown in liquid culture in the presence of cytokines able to induce differentiation versus the three main lineages. Doxorubicin, carboplatin and topotecan served as reference drugs to investigate the accuracy of the technique. The three drugs mimick the effects reported in vivo. Doxorubicin and carboplatin produce a specific effect toward erythropoietic and thrombopoietic lineages, respectively, and topotecan a three-lineage toxicity. An advantage of the technique is the possibility to further investigate myelotoxicity. Here, we assessed differentiation markers in CD34+ cells to evaluate if the three drug treatments can affect the process of differentiation. Data show that the drug treatments were unable to modulate the expression of the selected differentiation markers in the surviving population. We propose this method as an innovative tool to score the myelotoxic effect of compounds in the first steps of drug development to further develop those compounds with the best ratio between activity and myelotoxic effects. Moreover, the fact that the method is performed in liquid phase allows its optimisation in a conventional "high throughput system".

Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis.

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)

Alpha 2,3-specific desialylation of human red cells: effect on the autoantigens of the Pr, Sa and Sia-l1, -b1, -lb1 series.

BACKGROUND AND OBJECTIVES: Pr1,2,3, PrM, Sa and Sia-l1, -b1, -lb1 are sialic acid (NeuNAc)-dependent antigens recognized by human cold agglutinins. Pr and Sa antigens are the O-glycans of glycophorins containing alpha 2,3NeuNAc (to galactose) and/or alpha 2,6NeuNAc (to galactosamine). The antigens of the Sia-l1, -b1, -lb1 complex are gangliosides that may carry alpha 2,3NeuNAc (to galactose) and/or alpha 2,8NeuNAc (to NeuNAc). We studied the NeuNAc groups involved in the antigens. MATERIALS AND METHODS: From 74 sera with cold agglutinins against NeuNAc-dependent antigens, anti-T-free preparations were made and tested against human red cells, treated with an alpha 2,3-specific recombinant sialidase. RESULTS: Most (51/62) Pr antigens use alpha 2,3NeuNAc, some (8/62) use alpha 2,6NeuNAc and a few (3/62) use both sialyl groups as immunodominant components on glycophorins. The immunodominant component of Sa and Sia-l1, -b1, -lb1 determinants was alpha 2,3NeuNAc in all cases. CONCLUSION: The red cell target structures for cold agglutinins against NeuNAc-dependent antigens have been identified. We propose a Pr nomenclature to reflect this. The binding of anti-Pr to gangliosides may be the basis for anti-Pr-induced peripheral neuropathy.

In vitro proliferation and differentiation of erythroid progenitors of cord blood.

Stem cell factor (SCF) is known to synergize with erythropoietin (EPO) for erythropoiesis in vitro. Clonogenic assay and suspension culture were used to assess the effect of EPO alone or its combination with SCF on the proliferation and differentiation of erythroid progenitors of cord blood. Colony formation, increase in cell count, and cell cycling status for the proliferation as well as expression of Glycophorin A (Gly A) and hemoglobinization as the marker of differentiation were determined with each stimulation. The cell cycle status of the cells in suspension cultures was determined using FACScan after labeling of cells with propidium iodide. Expression of Gly A and degree of hemoglobinization were determined by FACScan and spectrophotometer on the cells plucked from colonies in semisolid culture. Larger increases in cell counts in suspension culture were observed with EPO + SCF after 12 days of inoculation than with EPO alone. Mean doubling time was 14.2 h with EPO + SCF and 22.7 h with EPO alone. The proportion of cells in S and G2 + M phase in day 14 suspension culture was 48% with EPO + SCF and 43% with EPO alone (no significant difference). Mean colony counts per 10(5) nonadherent mononuclear cells were 76 +/- 14 with EPO + SCF and 51 +/- 15 with EPO at day 14 (p < 0.05). The number of macroscopic colonies with > 0.5 mm diameter was 10.7 +/- 1.2 with EPO + SCF and 0.3 +/- 0.5 with EPO (p < 0.05). Percent of Gly A+ cells was 75% for both EPO + SCF and EPO colonies at day 14. Hemoglobin concentration/10(5) cells at day 14 was 0.70 +/- 0.17 microgram with EPO + SCF, and 1.16 +/- 0.32 micrograms with EPO alone (p < 0.05). In conclusion, SCF in the combination with EPO showed a synergistic effect for erythroid proliferation in colony number as well as colony size derived from cord blood, while SCF with EPO decreased hemoglobin synthesis but not Gly A expression at day 14.

Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid.

The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.

Red blood cell glycophorins as B and T-cell antigens in canine autoimmune haemolytic anaemia.

Pathogenic autoantibodies from two dogs with autoimmune haemolytic anaemia (AIHA) were shown to react with glycophorin from the canine red blood cell (RBC) membrane. Autoantibodies in both cases bound to purified glycophorin in enzyme-linked immunosorbent assays (ELISAs), and the major autoantigen immunoprecipitated by the antibodies corresponded in apparent molecular mass with glycophorin. Furthermore, neuraminidase treatment of the precipitated antigen, or of canine glycophorin, resulted in identical changes in apparent molecular mass in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Such removal of sialic acid from glycophorins was demonstrated to cause shifts in SDS-PAGE migration that are unique among RBC membrane proteins. In two further cases of AIHA, where autoantibodies did not immunoprecipitate the glycophorin pattern, ELISAs revealed that RBC-reactive IgG was present in serum and RBC elutes, but that these antibodies failed to bind to canine glycophorin. Thus, we consider that autoantibodies specific for glycophorin are present in some, but not all, dogs with AIHA. T-cells from a case of AIHA proliferated in vitro in response to autologous RBC, or to multiple RBC membrane components fractionated by SDS-PAGE. Three fractions, corresponding to major glycophorins, to the RBC anion channel band 3, and to spectrin from the membrane skeleton, were stimulatory. In contrast, T-cells from healthy dogs failed to respond to RBC, or to any blot fractions with the exception, in one animal, of the fraction bearing spectrin. It is suggested that activation of autoreactive T-cells with multiple specificities may be necessary to provide sufficient help for pathogenic autoantibody production.

Degradation of human erythrocyte surface components by human neutrophil elastase and cathepsin G: preferential digestion of glycophorins.

Human erythrocytes treated with purified human neutrophil elastase (HNE) or cathepsin G (CathG) were analysed by serological methods and by SDS-polyacrylamide gel electrophoresis followed by staining or immunoblotting with monoclonal antibodies. Both enzymes digested exhaustively glycophorins A, B and C, and HNE caused a partial digestion of band 3 protein. The degradation of other membrane proteins was not detectable by the methods used. Immunoblotting with the use of monoclonal antibodies against the defined epitopes of glycophorin A showed that HNE and CathG hydrolysed distinct peptide bonds in this antigen. The antibody PEP80, specific for the epitope in the cytoplasmic fragment of glycophorin A, gave patterns of bands which were characteristic for each of the two proteases. These bands could be distinctly identified in erythrocyte membrane samples containing only few percent of digested glycophorins. Therefore, the immunoblotting with this antibody may be useful as a sensitive assay for detecting the action of neutrophil proteases on red blood cells.

The effect of glycosylation trimming enzyme inhibitors on monoclonal antibody recognition of alpha-sialoglycoprotein epitopes.

The human erythrocyte membrane contains four sialoglycoproteins, denoted alpha, beta, gamma and delta (also known as glycophorins A, C, D and B respectively), of which alpha-sialoglycoprotein (alpha-SGP) is the most predominant species. The extracellular portion of alpha-SGP is heavily glycosylated with approximately 15 O-linked carbohydrate side-chains and a single N-linked group. We have used inhibitors of carbohydrate trimming enzymes to investigate the contribution of this single N-glycan moiety towards the recognition of a range of antibody binding sites on alpha-SGP. Two erythromyeloid cell lines, K562 and HEL, were cultured in the presence of these inhibitors and altered binding of antibodies to epitopes adjacent to the N-glycan was observed. Digoxigenin-coupled lectins were used to stain cytocentrifuge preparations and Western blots of cell lysates in order to confirm that modification of N-linked carbohydrate side-chains had been achieved. We suggest that the N-glycan side chain of alpha-SGP has a role in conferring conformational stability upon epitopes which lie in its vicinity.