Cloning and expression of an anti-LDL(-) single-chain variable fragment,and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr^-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr^-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).     

Characterization of two galactosidases extracted from wheat germ with a hydroalcoholic solvent.

Alpha- and beta-D-galactosidases were characterized from a hydroalcoholic extract of wheat germ (Triticum vulgare). Kinetic constants (Vmax and KM) and the optimal pHs for the hydrolysis of p-nitrophenyl galactopyranosides by both enzymes were determined. These enzymes presented a high stability in hydroalcoholic medium and were inhibited by iodoacetamide and sodium p-hydroxy-mercuribenzoate.     

New synthesis of immunogenic N6-isopent-2-enyladenosine-protein conjugate. Preparation, purification, and specificity of related antibodies.

An original procedure which preserves the structure of the sugar ring is described to link a plant hormone as N6-isopent-2-enyladenosine [( 9R]iP) onto a protein carrier to prepare a more specific immunogen. This cytokinin is bound to bovine serum albumin (BSA) and ovalbumin by a five-step procedure. These [9R]iP-protein conjugates have a maximal absorption at 269 nm and show molar ratios of hormone bound to proteins in the range of 12:1 and 18:1 for BSA and ovalbumin, respectively. Polyclonal antibodies were raised in rabbits against [9R]iP-BSA and were purified by affinity chromatography. Titers and specificity of the antisera and purified antibodies were determined by ELISA and RIA. These antibodies are highly specific for [9R]iP and do not cross-react with zeatin and ribosylzeatin. An immunoaffinity matrix was prepared with a capacity of 1 microgram of [9R]iP/mL of gel.     

The use of metallochromic Ca indicators in skeletal muscle

Absorbance signals recorded with metallochromic indicators in skeletal muscle fibers show rapid time courses that probably closely track the fast kinetic process of Ca++ release and retrapping by the sarcoplasmic reticulum. However, the formation of more than one complex in cuvette calibrations, both for Arsenazo III (ArIII) and Antipyrylazo III (ApIII), suggest that care needs to be taken in the deconvolution of in vivo absorbance signals. Since the kinetic rate constants have not yet been obtained for these probes, attempts to deconvolute absorbance signals should be considered approximate. The evidence suggesting that more than one complex is formed during a skeletal muscle transient with ArIII is more compelling than for the case of ApIII. The differences between the ArIII and ApIII signals may not be readily explained assuming 1:1 dye:Ca complexation and kinetic differences between the probes. Competition for Ca++ with cell Ca buffers and/or multiple complex formation by at least one of these probes needs to be invoked. Based on a simple model to simulate the behavior of the Ca signals in muscle, it may be suggested that an ApIII-like probe would more closely track pCa changes in the fiber than would an ArIII-like probe, which would show more interference with intracellular buffers; an even higher affinity probe would tend to sense the total release of Ca by the SR.     

Evolutionary Origins of a Bioactive Peptide Buried within Preproalbumin

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1’s additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.