Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.

Partially folded states of the cytolytic protein sticholysin II.

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.

Indice glucemico e insulinemico de seis frutas Venezolanas / Glycemic and insulinemic index of six Venezuelan fruits

La clasificación de carbohidratos en simples y complejos no predice su efecto sobre la glucosa o insulina sanguínea. El I.G de un alimento no puede ser inferido por el contenido de sus macronutrientes de forma aislada. Los índices glucémicos e insulinémicos, así como la respuestas de glucemia e insulinemia de las frutas estudiadas nos muestran que cantidades similares de carbohidratos en alimentos no necesariamente estimulan la secreción de insulina de una misma manera. Por lo tanto, el uso de distintas frutas en la dieta de los sujetos que presenten hipertrigliceridemia y otros trastornos metabólicos, debe hacerse de acuerdo al efecto de éstas sobre la glucemia e insulinemia postprandial.

Two-dimensional crystallization on lipid monolayers and three-dimensional structure of sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus.

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.

Overproduction in Escherichia coli and purification of the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus.

The cDNA coding for the cytolytic toxins sticholysin I and sticholysin II from the sea anemone Stichodactyla helianthus has been isolated, cloned in pUC18, and sequenced. A 6His-tagged version of sticholysin II has been overproduced in Escherichia coli and purified to homogeneity in milligram amounts. Conformational and functional analyses of recombinant sticholysin II do not reveal any significant difference when compared to the natural cytolysin.

Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein.

Sticholysin II (Stn-II) is a pore-forming cytolysin. Stn-II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer-tetramer equilibrium with an association constant K4c of 10(9) M(-3). The electrophoretic pattern of Stn-II treated with different cross-linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross-linking data.

Oligomerization of the cytotoxin alpha-sarcin associated with phospholipid membranes.

alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by alpha-sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.

Mechanism of the leakage induced on lipid model membranes by the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus.

A potent hemolytic polypeptide, sticholysin II, has been purified to homogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is present in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, coencapsulated with its quencher N,N'-p-xylenebispyridinium bromide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing 20-25% cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being observed for 20-30% sphingomyelin molar content. The extent of the leakage is dependent on the molecular mass of the vesicle entrapped solutes in the range 445-960 Da. This suggests the involvement of a pore of about 1 nm in diameter based on the limiting size observed for the leakage of the different solutes. Oligomerization of the protein is apparently involved in the membrane permeabilization, based on the kinetic analysis of the leakage process which is shown to proceed through an all-or-none mechanism.

Relations between fasting serum insulin, glucose, and dihydroepiandrosterone-sulfate concentrations in obese patients with hypertension: short-term effects of antihypertensive drugs.

A randomized, single-blind, placebo-controlled study was conducted in 82 obese patients with mild to moderate essential hypertension, to determine the incidence of hyperinsulinemia, the relations between fasting insulin and dihydroepiandrosterone-sulfate (DHEA-S) levels, and the short-term effects of antihypertensives on DHEA-S and insulin serum concentrations. Increased insulin/glucose ratios (IGR) suggestive of insulin resistance were found in half of our patients. Hyperinsulinemic and normoinsulinemic obese patients with hypertension had comparable fasting glucose and DHEA-S concentrations and comparable blood pressure (BP) levels. Thus no relations were found between fasting insulin and DHEA-S levels. Fasting hyperinsulinemia was found in only half of the obese subjects with hypertension, suggesting that not all obese patients with hypertension are at the same high cardiovascular risk. Short-term treatment with captopril, prazosin, verapamil, atenolol, or hydrochlorothiazide (HCTZ) reduced BP; greater BP reduction was observed with drugs with vasodilatory effects. Captopril, prazosin, and verapamil reduced fasting insulin levels, whereas atenolol and hydrochlorothiazide did not. The former drugs reduced fasting insulin levels that were either within normal limits or in the hyperinsulinemic range. None of the drug treatments produced significant increases in serum DHEA-S concentrations, although some of them considerably reduced fasting insulin levels. No relations between insulin and DHEA-S levels were observed either at baseline or at the end of the antihypertensive treatment. The BP reduction resulting from the peripheral vasodilation may explain the insulin-reducing action of captopril, verapamil, and prazosin. These results further emphasize the large heterogeneity present in the pathophysiologic mechanisms operating in obesity and hypertension.