Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen

Shrimp is a common cause of seafood hypersensitivity. To study the mechanism of seafood hypersensitivity at the molecular level, we have determined the primary structure of the major heat-stable allergen of shrimp by cloning, expression, nucleotide sequencing, and amino acid sequence determination of an IgE-reactive cDNA clone, Met e I, isolated from a Metapenaeus ensis expression library in lambda gt 11. We first constructed a cDNA library from the shrimp M. ensis in lambda gt 11. We then screened the library with sera from patients with hypersensitivity reactions to shrimp and identified a positive IgE-reactive clone, designated as Met e I. This cDNA was purified to homogeneity and subsequently expressed in the plasmid pGEX. Serum antibodies from patients with shrimp allergy demonstrated positive IgE reactivity by immunoblotting to a protein encoded by the clone Met e I; sera from nonallergic control subjects were not reactive. The nucleotide sequence of this cDNA clone revealed an open reading frame of 281 amino acid residues, coding for a protein of 34 kd. Comparison of the Met e I amino acid sequence with the Genbank database showed that Met e I is highly homologous to multiple isoforms of tropomyosin.     

The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence

Cytokines such as interferon-gamma (IFN-gamma), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-gamma or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-gamma is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95-132 of IFN-gamma (IFN-gamma(95-132)) containing the polybasic sequence 126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence 126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-gamma(95-132) but not the peptide IFN-gamma(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-gamma(95-132). This observation suggests that the NLS in IFN-gamma may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-gamma, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-gamma(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-gamma was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-gamma in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-gamma is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-gamma.     

Measurement of the beta-sheet-forming propensities of amino acids

Several model systems have been used to evaluate the alpha-helical propensities of different amino acids. In contrast, experimental quantitation of beta-sheet preferences has been addressed in only one model system, a zinc-finger peptide. Here we measure the relative propensity for beta-sheet formation of the twenty naturally occurring amino acids in a variant of the small, monomeric, beta-sheet-rich, IgG-binding domain from protein G. Amino-acid substitutions were made at a guest site on the solvent-exposed surface of the beta-sheet. Several criteria were used to establish that the mutations did not cause significant structural changes: binding to the Fc domain of IgG, calorimetric unfolding and NMR spectroscopy. Characterization of the terminal stabilities of these proteins leads to a thermodynamic scale for beta-sheet propensities that spans a range of approximately 2 kcal mol-1 for the naturally occurring amino acids, excluding proline. The magnitude of the differences suggests that beta-sheet preferences can be important determinants of protein stability.     

Mature T cell reactivity altered by peptide agonist that induces positive selection

Recent studies have investigated how defined peptides influence T cell development. Using a T cell receptor-transgenic beta2-microglobulin-deficient model, we have examined T cell maturation in fetal thymic organ cultures in the presence of various peptides containing single-alanine substitutions of the strong peptide agonist, p33. Cocultivation with the peptide A4Y, which contains an altered T cell contact residue, resulted in efficient positive selection. Several in vitro assays demonstrated that A4Y was a moderate agonist relative to p33. Although A4Y promoted positive selection over a wide concentration range, high doses of this peptide could not induce clonal deletion. Thymocytes maturing in the presence of A4Y were no longer able to respond to A4Y, but could proliferate against p33. These studies demonstrate that (a) peptides that induce efficient positive selection at high concentrations are not exclusively antagonists; (b) some agonists do not promote clonal deletion; (c) positive selection requires a unique T cell receptor-peptide-major histocompatibility complex interaction; and (d) interactions with selecting peptides during T cell ontogeny may define the functional reactivity of mature T cells.     

Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs

Combinations of EDTA-Tris and two aminoglycoside antibiotics (amikacin and neomycin) were tested for synergistic activities against the microorganisms associated with otitis externa in dogs and for the solutions' stability over time. Synergistic activity was observed when EDTA-Tris plus amikacin and EDTA-Tris plus neomycin were tested against Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli, but not against Candida albicans. Stability studies over a 3-month period indicated that the test solutions were stable at room temperature and that their antimicrobial activity was maintained.     

Riboflavin-mediated delivery of a macromolecule into cultured human cells

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine-sensitive increase of cell hydration by 6.5% +/- 1.2% (n = 3) and a transient phentolamine-sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2alpha, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (50 micromol/L). Single-cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and-consecutively-osmolyte content are modulated by hepatic nerves via an alpha-adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve-dependent alpha-adrenergic activation, whereas 3) KC and SEC probably do not express alpha-adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.