The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.     

Dual compartment (bicameral) culture: role of basement membrane in epithelial differentiation

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40–60 µ in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to ³H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteens are secreted in a polarized manner. In another study, hormone- stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrate decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity. This bicameral culture system could serve as an important model system to examine various functions of Sertoli cells including interactions of Sertoli cells with germ, Leydig, and myoid cells.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

Ion transport and regulation ofTetrahymena pyriformis in isotonic and hypotonic media

Summary The ion and volume regulatory mechanisms ofTetrahymena pyriformis were studied in normal or hypotonic nutrient media and in isotonic inorganic media with different Na/K ratios, in conjunction with the effects of a general metabolic inhibitor (low temperature) and a specific inhibitor (iodoacetate). For K two mechanisms of active influx were found: The first is sensitive to IAc and seems to be the basic mechanism for the maintenance of the K_i/K_o gradient. The second is sensitive to cooling and related to the function of the contractile vacuole; it is also responsible for the high intracellular levels of K. The passive K efflux seems to be a basic factor for volume regulation, together with the contractile vacuole. It increases in hypotonic media and this seems to be related to structural changes of the membranes occurring in hypotonic media. For Na two mechanisms of active transport were also found: One for active Na efflux with highK _m, which is associated with the contractile vacuole and another, for active Na influx with lowK _m, which is inhibited by high levels of intracellular K.The electrochemical potentials of Na and K and the membrane potential (Cl Nernst potential) were also studied in isotonic inorganic media. The membrane is negatively polarized, except if Na_o<5 mM when it becomes positive. In normal conditions, Na is transported outwards actively and leaks passively, while K is transported inwards actively and leaks 56 times more rapidly than Na ions.A model for the overall transport and regulation of ions inTetrahymena is proposed.Abbreviations IAc iodoacetate - PCV packed cell volume - Na _i,K _i,Cl intracellular concentrations ofNa ⁺,K ⁺,Cl ^–, respectively - Na _o,K _o, Cl_o extracellular concentrations of Na⁺, K⁺, Cl^–, respectively - DR distribution ratio - HyN hypotonic nutrient medium - IsN isotonic nutrient medium - HyS IsS hypotonic, and isotonic salt medium, respectively     

The anti-platelet approach targeting the fibrinogen ligand of the GPIIB/IIIa receptor.

Activation of the platelet surface receptor GPIIb/IIIa is the final pathway of platelet aggregation, regardless of the initiating stimulus. RGD analogues, peptidomimetics and monoclonal antibodies to GPIIb/IIIa have been developed targeting the blockage of the receptor and inhibition of the fibrinogen binding. However, the intrinsic activating effect of GPIIb/IIIa blockers is widely discussed as one potential contributing factor for the disappointing outcome of trials with GPIIb/IIIa inhibitors. An alternative method for thrombus prevention could be the use of specific fibrinogen blockers since they will act at the final step of the platelet aggregation and are expected to leave the receptor unaffected. To achieve this target the design of the fibrinogen ligands could be based on (i) sequences derived from GPIIb/IIIa ligand binding sites, and (ii) sequences complementary to RGD and/or to fibrinogen gamma-chain. The available information, which could be used as a starting point for developing potent fibrinogen ligands, is reviewed.     

Phosphono-sphingomyelins. I: Synthesis of ceramide (trimethyl) aminoethyl phosphonate

The synthesis of the phosphono-analogue of sphingomyelin is described. The N-acyl-D-erythro-sphingosyl-1-(N,N,N-trimethyl-2-aminoethyl) phosphonate was obtained by phosphonylation of N-acyl-3-O-benzoyl-D-erythro-sphingosine with (2-bromoethyl)phosphonic acid chloride and triethylamine, subsequent quaternisation with anhydrous trimethylamine and benzene at 55–60°C for four days, and finally, consecutive removal of the protective group by mild alkaline hydrolysis. Comparison of the CD spectra of both, natural sphingomyelin and its phosphono-analogue, confirmed that their structures and configurations were identical.     

A rapid and efficient method for the synthesis of selectively S-Trt or S-Mmt protected Cys-containing peptides

Selective removal of protecting groups under different cleavage mechanisms could be an asset in peptide synthesis, since it provides the feasibility to incorporate different functional groups in similar reactive centres. However, selective protection/deprotection of orthogonal protecting groups in peptides is still challenging, especially for Cys-containing peptides, where protection of the cysteine side-chain is mandatory since the nucleophilic thiol can be otherwise alkylated, acylated or oxidized. Herein, we established a protocol for the synthesis of Cys-selective S-Trt or S-Mmt protected Cys-containing peptides, in a rapid way. This was achieved by, simply fine-tuning the carbocation scavenger in the final acidolytic release of the peptide from the solid support in the classic SPPS.