Ng Peptide Research Articles

Synthesis of mono- and bifunctional peptide–dextran conjugates for the immobilization of peptide antigens on ELISA plates: properties and application

Dextran has been used as a carrier molecule for the synthesis of monofunctional peptide–dextran conjugates. The immunodetection of such carrier immobilized peptides on ELISA plates was compared to that of peptides adsorbed directly to immunoplates. The main features observed with peptide–dextran conjugates were as follows: only small amounts of peptide (1–2 mg) were necessary for coupling via α- or ϵ-amino groups to NaIO 4-activated dextran (4 mg); the coupling yield was up to 68%; an amino acid analysis of the conjugate enabled the amount of carrier immobilized peptide to be calculated; an estimated 15–17 peptides were bound per dextran molecule (MW 73,500); using a carbohydrate as carrier reduces the possibility of non-specific interactions because no hydrophobic or ionic sites and no protein-like epitopes exist on the carrier apart from the peptide ligand. It can be assumed that some peptide ligands provide the forces for an interaction with the plate surface whereas others remain free for the interaction with the antibody. Thus, the detection with monoclonal anti-peptide antibodies allowed peptide–dextran conjugates to be used at coating concentrations of 1–3 nM peptide, corresponding to 0.6–2.6 ng peptide–dextran per well. In contrast, concentrations of 150–500 nM were required for coating with peptides. The applicability of monofunctional peptide–dextran conjugates was demonstrated by investigating the titer and specificity of a polyclonal anti-peptide serum developed against human gastrointestinal glutathione peroxidase. The introduction of biotin as a second ligand of the dextran conjugate permitted its capture on streptavidin coated plates. This synthesis of bifunctional peptide–biotin–dextran conjugates opens up additional possibilities for applications.

Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts.

The resistance to cadmium ions (Cd-resistance) and possible formation of cadmium-binding complexes were examined in eight different wild-type yeasts. Saccharomyces exiguus, Pichia farinosa, Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibited partial Cd-resistance, as compared to the Cd-resistant strain 301N and the Cu-resistant but Cd-sensitive strain X2180-1B of Saccharomyces cerevisiae. Saccharomyces carlsbergensis, Pichia mogii, Zygosaccharomyces rouxii and Kluyveromyces lactis were all Cd-sensitive. The partially Cd-sensitive species, with the exception of S. exiguus, accumulated Cd2+ ions in the cytoplasmic fraction to varying extents. This fraction from S. octosporus included a Cd-binding complex that contained (gamma EC)nG peptides known as cadystins or phytochelatins, while P. farinosa and T. delbrueckii synthesized Cd-binding proteins that were similar to the Cd-metallothionein produced by S. cerevisiae 301N in terms of molecular weight and amino acid composition. These results suggest that such cytoplasmic molecules play a role in the Cd-tolerance of the above three species of yeast. S. exiguus retained most cadmium in the cell wall fraction and no Cd-binding complex was found in the cytoplasm, an indication of the important role of the cell wall in its Cd-tolerance. Different modes of binding of Cd2+ ions appear to be involved in the Cd-resistance of wild-type yeasts and fungi.

Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata

Cultures of Candida glabrata treated with CdCl 2 form intracellular Cd(II) complexes that evolve with the time of culturing. Initially, glutathione (γECG) appears to be the major buffering component. One type of Cd(II)-glutathione complex exists as a cadmium:sulfide (CdS) crystallite coated with glutathione. A time dependent change in the coating of the CdS particles occurs with a decrease in the (γECG) content and a corresponding increase in the abundance of (γEC)nG peptides with (γEC) 2G becoming the predominant peptide. The desGly variant (γEC) 2 appears in significant concentration only in late cultures. The evolution in isopeptide coating appears to be dependent on the sulfide content of the CdS particles. Cellular conditions that enhance the generation of sulfide ions facilitate the conversion from γECG to (γEC) 2G.

Metal ion resistance in fungi: Molecular mechanisms and their regulated expression

One stress response in cells is the ability to survive in an environment containing excessive concentrations of metal ions. This paper reviews current knowledge about cellular and molecular mechanisms involved in the response and adaptation of various fungal species to metal stress. Most cells contain a repertoire of mechanisms to maintain metal homeostasis and prevent metal toxicity. Roles played by glutathione, related (gamma-EC)nG peptides, metallothionein-like polypeptides, and sulfide ions are discussed. In response to cellular metal stress, the biosynthesis of some of these molecules are metalloregulated via intracellular metal sensors. The identify of the metal sensors and the role of metal ions in the regulation of biosynthesis of metallothionein and (gamma-EC)nG peptides are subjects of much current attention and are discussed herein.

Hypothalamic-pituitary-testicular function in rats after supraphysiological doses of a highly active LRH analogue (buserelin).

Abstract. Male pre-pubertal rats (60 g) were treated with the LRH analogue, [D-Ser(But)6]LRH(1—9)-nonapeptide-ethylamide (buserelin, Hoe 766), during 4 weeks by daily sc injections of 5, 50 or 500 ng peptide (group I, II and III). At the end of treatment, hypothalamic LRH content and arylamidase activity (LRH degrading enzyme) were not changed. Pituitary arylamidase activity was reduced, but the pituitary LRH receptors (tested by analogue binding in vitro) were not diminished. Pituitary accumulation of [125I]buserelin 60 min after iv injection was not modified and organ distribution in liver and kidney was unchanged. Pituitary responsiveness to the analogue was reduced at the highest dose, but there was significant LH-release at all three dose levels. Testosterone production in vitro (stimulated by hCG) was unaltered in group I and dramatically reduced in group II and III. Testicular testosterone content and hCG binding by testes homogenates were dose-dependently reduced. Histology of the testes after 4 weeks treatment showed minimal impairment of spermatogenesis at the highest dose, whereas the epididymis was almost devoid of sperm. The results indicate, that low dose treatment with a highly active LRH analogue, buserelin, does not interfere with pituitary responsiveness (LRH receptors and LH-release) and testicular function (testosterone production, testosterone content, LH-receptor level). At higher doses, pituitary and testicular responsiveness are dose-dependently inhibited. At the pituitary level, LRH receptors were not reduced. The antifertility effect of supraphysiological doses at the testicular level is explained by an LH-dependent loss of LH-receptors.