TCR-mediated activation of allergen-specific CD45RO(+) memory T lymphocytes results in down-regulation of cell-surface CXCR4 expression and a strongly reduced capacity to migrate in response to stromal cell-derived factor-1. (1/346)

The selective migration of functional T(h) lymphocyte subsets with different cytokine production profiles into inflamed tissue is likely to depend on the state of activation of the cells, as well as on the differential expression of various adhesion molecules and chemokine receptors. In this study, we have analyzed the effect of allergen-specific activation on the expression of the chemokine receptor CXCR4 on T lymphocytes. We show that stimulation of peripheral blood mononuclear cells from atopic patients with the allergen Der p results in down-regulation of CXCR4 surface expression on Der p-activated CD25(+)CD45RO(+) antigen-specific memory cells which was caused by a decrease in CXCR4 gene transcription and did not seem to be mediated by endogenous cytokines, such as IFN-gamma. In contrast, however, CXCR4 surface expression was enhanced on naive CD25(-)CD45RO(-) and resting CD25(-)CD45RO(+) memory T cells, as a result of the presence of endogenous IL-4, most likely produced by Der p-activated memory T cells. Antigen-specific CD25(+)CD45RO(+) T lymphocytes, purified 7 days after stimulation with Der p, had a strongly reduced capacity to migrate in response to stimulation with stromal cell-derived factor (SDF)-1, the ligand for CXCR4. Together, these results suggest that differential expression of CXCR4 on activated and resting T cells is due to the counteracting effects of TCR-mediated down-regulation and IL-4-mediated up-regulation of this chemokine receptor respectively, and furthermore indicate that antigen-activated memory T cells are unlikely to migrate into inflamed tissue in response to SDF-1.  (+info)

Functional and structural diversities of C-reactive proteins present in horseshoe crab hemolymph plasma. (2/346)

Limulin, a sialic-acid-binding and phosphorylethanolamine-binding hemagglutinin in the hemolymph plasma of the American horseshoe crab (Limulus polyphemus), is a hemolytic C-reactive protein [Armstrong, P.B., Swarnakar, S., Srimal, S., Misquith, S., Hahn, E.A., Aimes, R. T. & Quigley, J.P. (1996) J. Biol. Chem. 271, 14717-14721]. We have now identified three types of C-reactive protein in the plasma of the Japanese horseshoe crab (Tachypleus tridentatus), based on different affinities against fetuin-agarose and phosphorylethanolamine-agarose determined by quantitative precipitin assays using fetuin and an artificial phosphorylethanolamine-protein conjugate. Partial amino acid sequences of the isolated C-reactive proteins identified homologous proteins which were named Tachypleus tridentatus CRP-1 (tCRP-1), tCRP-2 and tCRP-3, each of which possibly constitute isoprotein mixtures. tCRP-2 and tCRP-3, but not tCRP-1, agglutinated mammalian erythrocytes. tCRP-1, the most abundant C-reative protein in the plasma, exhibited the highest affinity to the phosphorylethanolamine-protein conjugate but lacked both sialic-acid-binding and hemolytic activities. tCRP-2 bound to both fetuin-agarose and phosphorylethanolamine-agarose, and exhibited Ca2+-dependent hemolytic and sialic-acid-binding activities, suggestive of limulin-like properties. Furthermore, tCRP-2 exhibited a higher affinity to colominic acid, a bacterial polysialic acid. By contrast, tCRP-3 shows stronger hemolytic, sialic-acid-binding and hemagglutinating activities than tCRP-2. tCRP-3 has no affinity to phosphorylethanolamine-agarose, phosphorylethanolamine-protein conjugate and colominic acid. This suggests tCRP-3 is a novel hemolytic C-reactive protein lacking a common characteristic of phosphorylethanolamine-agarose binding affinity. Twenty-two clones of tCRPs with different deduced amino acid sequences were obtained by PCR using oligonucleotide primers based on the N-terminal and C-terminal sequences of tCRPs and with templates including genomic DNA and cDNA of hemocytes or hepatopancreas derived from one individual. The translation products of the tCRP clones possess high molecular diversity which falls into three related groups, consistent with classification based on their biological activities. Only tCRP-3 contained a unique hydrophobic nonapeptide sequence that appears in the transmembrane domain of a major histocompatibility complex class I heavy chain of rainbow trout, suggesting the importance of the hydrophobic patch to the hemolytic activity of tCRP-3. The structural and functional diversities of tCRPs provide a good model for studying the properties of innate immunity in invertebrates, which survive without the benefit of acquired immunity.  (+info)

A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. (3/346)

Molting or ecdysis is the most fundamentally important process in arthropod life history, because shedding of the exoskeleton is an absolute prerequisite for growth and metamorphosis. Although the hormonal mechanisms driving ecdysis in insects have been studied extensively, nothing is known about these processes in crustaceans. During late premolt and during ecdysis in the crab Carcinus maenas, we observed a precise and reproducible surge in hemolymph hyperglycemic hormone (CHH) levels, which was over 100-fold greater than levels seen in intermolt animals. The source of this hormone surge was not from the eyestalk neurosecretory tissues but from previously undescribed endocrine cells (paraneurons), in defined areas of the foregut and hindgut. During premolt (the only time when CHH is expressed by these tissues), the gut is the largest endocrine tissue in the crab. The CHH surge, which is a result of an unusual, almost complete discharge of the contents of the gut endocrine cell, regulates water and ion uptake during molting, thus allowing the swelling necessary for successful ecdysis and the subsequent increase in size during postmolt. This study defines an endocrine brain/gut axis in the arthropods. We propose that the ionoregulatory process controlled by CHH may be common to arthropods, in that, for insects, a similar mechanism seems to be involved in antidiuresis. It also seems likely that a cascade of very precisely coordinated release of (neuro) hormones controls ecdysis.  (+info)

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata. (4/346)

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.  (+info)

Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site. (5/346)

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3' and 5' RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His(211) in this conserved motif was shown to be very important in catalysis by site-specific modification with (14)C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg(2+) and Ca(2+). The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.  (+info)

Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor. (6/346)

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.  (+info)

The underlying mechanism for the diversity of disulfide folding pathways. (7/346)

The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native- like structures.  (+info)

Synthetic endotoxin-binding peptides block endotoxin-triggered TNF-alpha production by macrophages in vitro and in vivo and prevent endotoxin-mediated toxic shock. (8/346)

Lipid A, the conserved portion of endotoxin, is the major mediator of septic shock; therefore, endotoxin-neutralizing molecules could have important clinical applications. Here we show that peptides derived from Limulus anti-LPS factor (LALF), bactericidal/permeability increasing protein (BPI) and endotoxin-binding protein, bind to lipid A and block the recombinant LALF/lipid A interaction in vitro. Because their neutralizing capacity in vitro as well as in vivo has been limited, we created hybrid peptides comprising two endotoxin-binding domains. The hybrid molecule LL-10-H-14, containing endotoxin-binding domains from LALF and endotoxin-binding protein, turned out to be the most active peptide within the series of peptides tested here to inhibit the CD14/lipid A interaction and is able in vitro to block the endotoxin-induced TNF-alpha release of murine macrophages up to 90%. Furthermore, LL-10-H-14 not only reduced peak serum levels of TNF-alpha of mice when preinjected but also reduced TNF-alpha levels when given 15 min after the endotoxin challenge. As compared with other peptides, only LL-10-H-14 is able to strongly decrease endotoxin-stimulated TNF-alpha release by human macrophage cell lines as well as by PBMC. Furthermore, the hybrid peptide is protective against endotoxin-provoked lethal shock. As such, LL-10-H-14 could have prophylactic and/or therapeutic properties in humans for the management of septic shock.  (+info)

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