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Abstract:The ab initio fragment molecular orbital (FMO) calculations were performed for the cAMP receptor protein (CRP) complexed with a cAMP and DNA duplex to elucidate their sequence-specific binding and the stability of the DNA duplex, as determined by analysis of their inter-and intramolecular interactions. Calculations were performed with the AMBER94 force field and at the HF and MP2 levels with several basis sets. The interfragment interaction energies (IFIEs) were analyzed for interactions of CRP-cAMP with each base pair, DNA duplex with each amino acid residue, and each base pair with each residue. In addition, base-base interactions were analyzed including hydrogen bonding and stacking of DNA. In the interaction between DNA and CRP-cAMP, there was a significant charge transfer (CT) from the DNA to CRP, and this CT interaction played an important role as well as the electrostatic interactions. It is necessary to apply a quantum mechanical approach beyond the "classical" force-field approach to describe the sequence specificity. In the DNA intramolecular interaction, the dispersion interactions dominated the stabilization of the base-pair stacking interactions. Strong, attractive 1,2-stacking interactions and weak, repulsive 1,3-stacking interactions were observed. Comparison of the intramolecular interactions of free and complexed DNA revealed that the base-pairing interactions were stronger, and the stacking interactions were weaker, in the complexed structure. Therefore, the DNA duplex stability appears to change due to both the electrostatic and the CT interactions that take place under conditions of DNA-CRP binding.

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The American horseshoe crab Limulus polyphemus contains a,-macroglobulin (a,M) in the hemolymph plasma and hemocytes. a,M from Lirnulus shows many of the typical characteristics of mammalian a,M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian a,M receptor. Additionally, Limulus rx, M has the unique property that it regulates the limulin-based hemolytic system of the plasma.A cDNA encoding Limulus a,M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian uZMs (28-29% identity) and contains common features found in mammalian a,Ms, a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian a,Ms, and it is structurally related to that of the human complement factor C8y chain, consistent with a role for Limulus a,M in host defense. The component sugar analysis of Limulus a,M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian u,M. However, unlike mammalian m2M, no sialic acid was detected in Limulus rx,M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of 0-linked sugar chains, which have not been found in mammalian n,M.Expression of a,M was detected in hemocyies, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against a,M indicated the presence of the cx,M in large granules. Trypsintreated Linzu/us a2M, but not the native a2M, displaced methylamine-treated human ' "51-u2M from the human cx,M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.Keyuvrds: u2-macroglobulin; horseshoe crab; invertebrate; cDNA.Proteins of the az-macroglobulin (a,M) family are abundant components in the plasma of mammals (Sottrup-Jensen, 1987, 1989 and arthropods (Armstrong et al., 1996;, comprising about 3% of the total plasma protein of humans (Ganrot and Schersten, 1967) and the third most abundant protein in the plasma of the American horseshoe crab, Linzulzcs polyphemus Ci,r.r.f,sl,o~zde,zc.e

This study was designed to determine seasonal changes in cytokines, soluble CD23 and specific IgE in the serum of patients with seasonal allergic rhinitis, and the effect of immunotherapy on these seasonal changes. Fifty-four patients with seasonal allergic rhinitis caused by Japanese cedar pollens were divided into a medication group and an immunotherapy group. The patients of the medication group were treated with nonsedating antihistamines alone during the pollen season. The patients of the immunotherapy group had been treated for variable periods (mean, 5.0 Ϯ 3.2 years) with immunotherapy using Japanese cedar pollen antigens. Serum samples were collected before and during the pollen season from each patient, to determine specific IgE, interleukin-4 (IL-4), interferon-g (IFN-g) and soluble CD23 levels in serum. A significant increase in specific IgE and IL-4 and a significant decrease in IFN-g were observed during the pollen season in the medication group. In contrast, in the immunotherapy group, none of specific IgE, IL-4 and IFN-g was significantly changed following natural exposure to pollens. However, these effects were not significant in patients undergoing immunotherapy for 3 or fewer years. Seasonal rates of increase in specific IgE and IL-4 differed significantly between good responders and poor responders to immunotherapy, but seasonal rates of decrease in IFN-g did not. A seasonal rate of increase in soluble CD23 was significantly correlated with a seasonal rate of increase in specific IgE, in both the medication and the immunotherapy groups. The seasonal rate of increase in soluble CD23 was significantly smaller in the good responders than in the poor responders to immunotherapy. In conclusion, pollen immunotherapy reduces the seasonal increase in specific IgE, IL-4 and soluble CD23 in serum, and in addition switches the seasonal preferential activation of Th-2 cells to reciprocal activation of Th-1 cells with treatment over several years. It is likely that the mechanisms responsible for the clinically beneficial effects of immunotherapy principally involve the modulation of Th-2 rather than Th-1 cytokines.

Some individuals with detectable levels of Japanese cedar (Criptomeria japonica) pollen-specific immunoglobulin (Ig)E in serum have no apparent nasal symptoms during the pollen season. The response of CD4+ T-helper (Th) cells to the pollen allergen might differ fundamentally between asymptomatic and symptomatic individuals who are already sensitized to the pollen. The aim of this study was to discern the possible differences in responses of peripheral blood mononuclear cells (PBMCs) to the pollen allergen between asymptomatic and symptomatic subjects who have been sensitized to the pollen. This study included 20 non-atopic healthy volunteers (non-atopic group) and 48 patients who had detectable levels of the pollen-specific IgE before the pollen season in 1997. In the review of nasal symptoms during the pollen season 1997, 24 patients had typical symptoms of seasonal allergic rhinitis (symptomatic group), and the remainder had no seasonal aggravation of nasal symptoms (asymptomatic group). Peripheral blood mononuclear cells (1.0 x 10(7) cells/well) were obtained from each individual during the pollen season and cultured in the absence or presence of 12.5 microg of Cry j 1 for 4 days. The concentrations of IgE, interleukin-5 (IL-5), and interferon-gamma (IFN-gamma) in the culture supernatants were measured. The levels of IgE produced by Cry j 1-stimulated PBMCs of the asymptomatic and symptomatic groups were significantly higher than those of the non-atopic group, but did not differ between the asymptomatic and symptomatic groups. The levels of IL-5 produced by Cry j 1-stimulated PBMCs did not differ significantly between the non-atopic group and the asymptomatic group, but the levels of IL-5 were significantly higher in the symptomatic group than in the asymptomatic group as well as the non-atopic group. The levels of IFN-gamma produced by Cry j 1-stimulated PBMCs did not differ significantly among the three groups. In conclusion, our study has suggested that Japanese cedar pollen-induced synthesis of IL-5, but not of IgE or IFN-gamma, is likely to be a key mechanism linked to the symptomatic episode of seasonal allergic rhinitis in individuals sensitized to the pollen.

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