肌肽棉酚对绵羊精子运动及超微结构的影响

肌肽促进绵羊精子运动增强,直线运动精子数目增多;棉酚则强烈抑制精子运动并使精子的运动方式发生改变;肌肽与棉酚混合处理精子,肌肽可不同程度抵消棉酚对精子产生的有害作用。但当棉酚完全抑制精子运动后,再加入肌肽则不能使精子恢复运动。电镜观察,肌肽对绵羊精子超微结构无任何不良影响;棉酚同造成精子严重损伤,导致生物膜系统损害、轴丝崩解、线粒体螺旋鞘紊乱等。本文通过肌肽,棉酚加入的时序不同,探讨两者对绵羊精子     

薄层色谱和高效液相色谱联用测定暗纹东方鲀肌肉中肌肽和谷胱甘肽的含量

通过薄层色谱(TLC)与高效液相色谱(HPLC)联用技术鉴定了暗纹东方鲀(Takifugu obscurus)肌肉中存在肌肽和谷胱甘肽,同时利用高效液相色谱法测定了肌肽和谷胱甘肽(GSH)的含量。薄层层析采用的展开剂为正丁醇:乙酸:水(4∶2∶1),层析板为硅胶板。高效液相色谱利用Kromasil C18反相柱分析,流动相为10%的纯乙腈和90%含有0.05%三氟乙酸的超纯水。结果表明:通过薄层色谱和高效液相色谱鉴定了暗纹东方鲀肌肉中肌肽和谷胱甘肽,其中暗纹东方鲀肌肉中肌肽含量约213μg/g(鲜重),还原性谷胱甘肽含量约211μg/g(鲜重)。本法样品无需衍生,操作简便,适合于暗纹东方鲀肌肉中肌肽和谷胱甘肽的测定。本文为生物体内肌肽和谷胱甘肽的研究提供借鉴意义。     

LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress in excitable tissues

A sensitive, selective, specific and rapid liquid chromatographic-electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination in skeletal muscle of the Michael adducts between 4-hydroxy-trans-2-nonenal (HNE), one of the most reactive lipid peroxidation-driven unsaturated aldehyde, and glutathione (GSH) and the endogenous histidine-containing dipeptides carnosine (CAR) and anserine (ANS), with the final aim to use conjugated adducts as specific and unequivocal markers of lipid peroxidation. Samples (skeletal muscle homogenates from male rats) were prepared by protein precipitation with 1 vol. of a HClO(4) solution (4.2%; w/v) containing H-Tyr-His-OH as internal standard. The supernatant, diluted (1:1, v/v) in mobile phase, was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9:1:0.01, v/v/v) at a flow rate of 0.2 ml/min, with a run time of 12 min. Detection was on a triple quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were in multiple reaction monitoring (MRM) mode using the following precursor-->product ion combinations: H-Tyr-His-OH (IS): m/z 319.2--> 156.5+301.6; GS-HNE: m/z 464.3--> 179.1+308.0; CAR-HNE: m/z 383.1--> 110.1+266.6; ANS-HNE: m/z 397.2--> 109.1+126.1. The method was validated over the concentration ranges 1.5-90 (GS-HNE) and 0.4-40 (CAR-HNE, ANS-HNE) nmoles/g wet tissue, and the LLOQ were 1.25 and 0.33 pmoles injected respectively. The intra- and inter-day precisions (CV%) were <7.38% (相似文献   

Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases

There are numerous publications describing the positive effects of carnosine (beta-alanyl-histidine) and anserine (beta-alanyl-1-N-methyl-histidine) on cell and organ function. Of special interest to us is the fact that these dipeptides act to retard and (in one instance) reverse non-enzymatic glycation. To date, the primary explanation for these anti-glycating effects has been the fact that carnosine and anserine can serve as alternative and competitive glycation targets, thereby protecting proteins from this deleterious process. In this paper, we document another mechanism by which these two peptides can retard or reverse glycation. The process involves decomposition of the very first intermediates of the non-enzymatic glycation cascade (aldosamines a.k.a. Schiff bases) by nucleophilic attack of carnosine and/or anserine on the preformed aldosamine such as glucosyl-lysine. If future research shows this reaction is to be physiologically important, this mechanism could explain some of the beneficial effects of carnosine and anserine as anti-glycating agents.     

Novel molecular approaches for improving enzymatic and nonenzymatic detoxification of 4-hydroxynonenal: toward the discovery of a novel class of bioactive compounds

4-Hydroxy-trans-2-nonenal (HNE), an α,β-unsaturated aldehyde generated endogenously by the radical-mediated peroxidation of ω-6 polyunsaturated fatty acids, is a bioactive molecule acting in several physiopathological mechanisms and most of its activity is due to the covalent modification of biomolecules. Although at low and physiological levels HNE acts as an endogenous signaling molecule, a growing bulk of evidence indicates that at high and toxic concentrations, HNE is involved in the onset and propagation of several human diseases. To get more conclusive evidence of HNE as a pathogenetic factor, a pharmacological tool able to inhibit the HNE-induced cellular response is required. Such compound is currently not available, although several molecular strategies have so far been reported with the aim of inhibiting HNE formation or catalyzing its removal. Although most of these are not selective, such strategies have been found to induce several biological responses and would merit further investigation. In this review the various strategies are reported and discussed together with their limits and potentials.     

Carnosine Release from Olfactory Bulb Synaptosomes Is Calcium-Dependent and Depolarization-Stimulated

Abstract: The dipeptide carnosine (β-alanyl-L-histidine) has been proposed as a neurotransmitter in the mammalian olfactory pathway. Therefore, the efflux of in vivo -synthesized [¹⁴C]carnosine from mouse olfactory bulb synaptosomes was investigated. Carnosine was found to be released from the olfactory bulb synaptosomes by two mechanisms. The first is a slow spontaneous process that is independent of depolarization. The rate of this release was doubled in the presence of 1 m M external carnosine. Release by the second mechanism was markedly stimulated in the presence of calcium by depolarization with either 60 m M K⁺ or 300 μ M veratridine. Omission of calcium abolished the stimulatory effect of both of these agents. Further, blockage of the veratridine-induced depolarization by tetrodotoxin also inhibited carnosine release. These results are consistent with the hypothesis that carnosine acts as a neurotransmitter in the mouse olfactory pathway.     

Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (β-alanyl-l-histidine), anserine (β-alanyl-π-methyl-l-histidine), and balenine (ophidine; β-alanyl-τ-methyl-l-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275 kDa. SDS-PAGE of the purified enzyme represented a band around 43 kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 °C, respectively. K_m values for β-alanine and π-methyl-l-histidine were 44 and 89 mM, respectively. The enzyme was greatly activated by Zn²⁺ and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38–62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.     

Carnosine Protects Against Aβ42-induced Neurotoxicity in Differentiated Rat PC12 Cells

(1) The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Aβ42-induced impairment in differentiated PC12 cells. (2) PC12 cells were exposed to Aβ42 (5 μM) for 24 h after carnosine (5 mM) applied for 18 h. Histamine receptor antagonists (diphenhydramine, zolantidine, thioperamide, clobenpropit) or histidine decarboxylase inhibitor (α-fluoromethylhistidine) were added 15 min before carnosine. Cell viability, glutamate release or cell surface expression of NMDA receptor was examined. (3) Aβ42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with carnosine ameliorated this impairment. This amelioration was reversed by the H₃ receptor antagonists thioperamide and clobenpropit, but not by either the H₁ receptor antagonist diphenhydramine or the H₂ receptor antagonist zolantidine. Further, α-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, also had no effect. In the presence of Aβ42, carnosine significantly decreased glutamate release and carnosine increased the surface expression of NMDA receptor. (4) These results indicate that the mechanism by which carnosine attenuates Aβ42-induced neurotoxicity is independent of the carnosine–histidine–histamine pathway, but may act through regulation of glutamate release and NMDA receptor trafficking.     

Carnosine as molecular probe for sensitive detection of Cu(II) ions using localized 1H NMR spectroscopy

Complex formation of carnosine (Csn) with Cu(II) is suspected to be of significant biochemical importance and can be detected by NMR via ion-induced paramagnetic relaxation of Csn signals. Here, we present quantification of the sensitivity achieved with localized (1)H NMR spectroscopy at physiological pH and high ligand-to-metal ratios. While characterizing the highly effective relaxation transfer onto a huge Csn pool due to fast ligand exchange, it is demonstrated that a metal-to-ligand ratio of approximately 100 ppm suffices to reduce Csn signals by approximately 50% in vitro, thus making the dipeptide a sensitive probe for such ions. Variation of the donor accessibility reveals that the paramagnetic effect is transferred onto a approximately 1370-fold donor abundance for a given ion concentration. A method is presented to characterize such effective ligand exchange relaxation transfer. These studies focus on the monomer formation since comparison with (1)H NMR data of human calf muscle demonstrates that the dimer complex is insignificant in vivo. Observed line broadening in living tissue yields an upper limit of ca. 195 ppm for the Csn-related copper concentration in human skeletal muscle.     

Carnosine Synthesis in Olfactory Tissue During Ontogeny: Effect of Exogenous β-Alanine

Carnosine has now been demonstrated by chemical analysis to be present in rat olfactory mucosa on day 16 of gestation. The tissue content of this dipeptide then increases progressively during fetal and postnatal life. Radioactive carnosine can be isolated from cultured embryonic rat olfactory mucosa incubated with [14C]beta-alanine as early as 13-14 days of gestation. The amount of incorporation also increases progressively with the initial age of the explant and with time in culture indicating in vitro maturation of the carnosine synthesis capability of olfactory tissue. To test whether the level of beta-alanine was limiting the synthesis of carnosine, we evaluated the effect of elevated beta-alanine levels on tissue carnosine content. Exogenous beta-alanine caused an increase in the tissue content of carnosine at several ages in vivo and in vitro. In adult animals this increase was observed in olfactory bulb, olfactory mucosa, and skeletal muscle. However, there was no associated alteration in carnosine synthetase activity. In addition, the different half-lives of carnosine in olfactory tissue and muscle seemed unaltered, arguing against any effect on degradative enzymes. Thus, tissue carnosine levels are regulated, at least in part, by substrate availability. The early appearance of carnosine synthetic capacity during prenatal development indicates that this enzyme activity should be a valuable aid in studying early events in olfactory neuron maturation.