Effects of elevated egg corticosterone levels on behavior, growth, and immunity of yellow-legged gull (Larus michahellis) chicks

Eggs of vertebrates contain steroid hormones of maternal origin that may influence offspring performance. Recently, it has been shown that glucocorticoids, which are the main hormones mediating the stress response in vertebrates, are transmitted from the mother to the egg in birds. In addition, mothers with experimentally elevated corticosterone levels lay eggs with larger concentrations of the hormone, which produce slow growing offspring with high activity of the hypothalamo-adrenal axis under acute stress. However, the effects and function of transfer of maternal corticosterone to the eggs are largely unknown. In the present study, we injected corticosterone in freshly laid eggs of yellow-legged gulls (Larus michahellis), thus increasing the concentration of the hormone within its natural range of variation, and analyzed the effect of manipulation on behavioral, morphological, and immune traits of the offspring in the wild. Eggs injected with corticosterone had similar hatching success to controls, but hatched later. Mass loss during incubation was greater for corticosterone-treated eggs, except for the last laid ones. Corticosterone injection reduced rate and loudness of late embryonic vocalizations and the intensity of chick begging display. Tonic immobility response, reflecting innate fearfulness, was unaffected by hormone treatment. Elevated egg corticosterone concentrations depressed T-cell-mediated immunity but had no detectable effects on humoral immune response to a novel antigen, viability at day 10, or growth. Present results suggest that egg corticosterone can affect the behavior and immunity of offspring in birds and disclose a mechanism mediating early maternal effects whereby stress experienced by females may negatively translate to offspring phenotypic quality.     

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography-tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.     

Novel and simple high-performance liquid chromatographic method for determination of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

We present here a high-performance liquid chromatographic method for the evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. The automated method was applied to fungal and mouse liver extracts and validated by the addition of mevastatin to the reaction mixture and by several intra- and inter-day assays. This method offers important advantages over those previously reported because no radiolabeled substrates or expensive techniques such as mass spectrometry are required, and the time of analysis is relatively short. Moreover, the method can be successfully applied to different biological samples; hence, it should be very useful in evaluating potential inhibitors of the HMG-CoA enzyme and investigating cholesterol metabolism, cell growth and differentiation processes.     

Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins

In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common intermediate in the folding of all members of the globin family, we have extensively studied a site-directed mutant of the myoglobin from Aplysia limacina, distantly related to the mammalian counterpart, in which one of the two tryptophans in the A-G-H cluster [i.e., Trp(H8)130] has been mutated to tyrosine. The results presented here show that this mutation destabilizes both the native state and the acid intermediate I(A) but exerts little or no effect on the thermally stable core of an intermediate species (called I(T)) peculiar to Aplysia apomyoglobin. Dynamic quenching of Trp emission by acrylamide provides information on the accessibility of the chromophores at the native and the intermediate states of wild-type and mutant Aplysia apomyoglobin, consistent with the thermodynamics. Our results agree well with those obtained for the corresponding topological position of apomyoglobin from sperm whale and clearly show that the H8 position is involved in the stabilization of the main intermediate in both apoproteins. This residue thus plays a role which is evolutionarily conserved in the globin family from invertebrates to mammals; our results support the contention that the A-G-H cluster is important in the folding pathway of different globins.     

Nucleotide excision repair defect influences lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent in the absence of base excision repair

Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, [1-methyl-4-[1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propane, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency. In the present work, we have explored the role of nucleotide excision repair (NER) in Me-lex-induced toxicity and mutagenicity since it is known that NER acts on abasic sites in vivo in yeast and in vitro assays. To accomplish this, we have deleted one of the genes essential for NER in yeast, namely, RAD14, both in the context of an otherwise DNA repair-proficient strain (Deltarad14) and in a BER-defective isogenic derivative lacking the MAG1 gene (Deltamag1rad14). Interestingly, no sensitivity to the treatment with Me-lex was conferred by the simple deletion of RAD14. However, a significant enhancement in toxicity and mutagenicity was observed when cells lacked both Rad14 and Mag1. The mutation spectrum induced by Me-lex in the Deltamag1rad14 strain is indistinguishable from that observed in the Deltaapn1/Deltaapn2 or in the Deltamag1 strains. The results indicate that in yeast NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity; however, the role of NER is appreciable only in a BER-defective background.     

beta-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor beta-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with beta-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from beta-arrestin activate any G-protein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.