Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats.

Adrenomedullin (AM) immunoreactivity and mRNA, in addition to a large number of specific AM-binding sites, exist in the rat spinal cord. However, no phenotype has been reported for AM in the spinal cord. Here, expression of c-fos in response to intrathecal (i.t.) administration of AM, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) was examined in the thoracic, lumbar and sacral regions of spinal cord in conscious rats. Two hours after i.t. administration of either CGRP (2.5 and 10 microg) or AM (10 microg), the number of c-Fos immunoreactive nuclei was increased in all the spinal regions examined in this study, with the highest increase observed in the superficial dorsal horn. Few cells with c-fos immunoreactivity were found in the spinal cord of rats 2 h after i.t. injection of either saline or PAMP. Effects of AM (10 microg) and CGRP (2.5 microg) on c-fos expression were blocked when rats were pretreated with 40 microg of intrathecal CGRP8-37 (CGRP1 receptor antagonist). Fos-like immunoreactivity induced by i.t. CGRP and/or AM were also significantly abolished by i.t. administration of the nitric oxide (NO) inhibitor, l-NAME, indicating that endogenous NO is a necessary intermediary in CGRP and AM induced c-fos expression in the rat spinal cord. In conclusion, AM induces c-fos expression in rat spinal cord when administered intrathecally, with the pattern being similar to those produced by i.t. CGRP. Effects of the two peptides are sensitive to CGRP8-37 and l-NAME.

Effect of opium smoking on concentrations of carcinoembryonic antigen and tissue polypeptide antigen.

Previous studies have related opium and its pyrolysates to the risk of developing certain cancers. The aim of this work was to evaluate the clinical usefulness of determining carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA) levels in habitual opium smokers. Serum CEA concentrations were measured in 128 opium smokers and in 44 controls of cigarette only smokers and 47 normal non-smokers by an EIA-based assay. TPA levels were also determined in serum and urine of a subgroup in the study population. The results indicated that serum CEA concentrations are higher in opium smokers than in healthy tobacco smokers (p = 0.004) and non-smokers (p = 0.001). The amount of opium used correlated with the serum CEA level (r = 0.276, p < 0.0001). The mean urine and serum TPA levels of the opium-addicted population were also higher than that of the non-smoking control group, but the differences were not statistically significant. We conclude that opium smoking is associated with elevated serum CEA levels. Therefore, for management of opium users with neoplastic diseases, increased levels of serum CEA should be viewed with caution to avoid misdiagnosis.

Crystal structure of a phage library-derived single-chain Fv fragment complexed with turkey egg-white lysozyme at 2.0 A resolution.

The three-dimensional structure of the single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme (TEL) has been determined to a nominal resolution of 2.0 A by X-ray diffraction. The scFv fragment 1F9 was derived from phage-display libraries in two steps and binds both hen and turkey egg-white lysozyme, although the level of binding affinity is two orders of magnitude greater for the turkey lysozyme. The comparison of the crystal structure with a model of the single-chain Fv fragment 1F9 in complex with hen egg-white lysozyme (HEL) reveals that in the latter a clash between Asp101 in lysozyme and Trp98 of the complementarity determining region H3 of the heavy chain variable domain occurs. This is the only explanation apparent from the crystal structure for the better binding of TEL compared to HEL. The binding site topology on the paratope is not simply a planar surface as is usually found in antibody-protein interfaces, but includes a cleft between the light chain variable domain and heavy chain variable domain large enough to accommodate a loop from the lysozyme. The scFv fragment 1F9 recognizes an epitope on TEL that differs from the three antigenic determinants recognized in other known crystal structures of monoclonal antibodies in complex with lysozyme.

Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain.

The 1,3-1,4-beta-glucanase from Bacillus macerans (wtGLU) and the 1, 4-beta-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 A resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Crystal structures and properties of de novo circularly permuted 1,3-1,4-beta-glucanases.

The 1,3-1,4-beta-glucanases from Bacillus macerans and Bacillus licheniformis, as well as related hybrid enzymes, are stable proteins comprised of one compact jellyroll domain. Their structures are studied in an effort to reveal the degree of redundancy to which the three-dimensional structure of protein domains is encoded by the amino acid sequence. For the hybrid 1,3-1,4-beta-glucanase H(A16-M), it could be shown recently that a circular permutation of the sequence giving rise to the variant cpA16M-59 is compatible with wildtype-like enzymatic activity and tertiary structure (Hahn et al., Proc. Natl. Acad. Sci. USA 91:10417-10421, 1994). Since the circular permutation yielding cpA16M-59 mimicks that found in the homologous enzyme from Fibrobacter succinogenes, the question arose whether de novo circular permutations, not guided by molecular evolution of the 1,3-1,4-beta-glucanases, could also produce proteins with native-like fold. The circularly permuted variants cpA16M-84, cpA16M-127, and cpA16M-154 were generated by PCR mutagenesis of the gene encoding H(A16-M), synthesized in Escherichia coli and shown to be active in beta-glucan hydrolysis. CpA16M-84 and cpA16M-127 were crystallized in space groups P2(1) and P1, respectively, and their crystal structures were determined at 1.80 and 2.07 A resolution. In both proteins the main parts of the beta-sheet structure remain unaffected by the circular permutation as is evident from a root-mean-square deviation of main chain atoms from the reference structure within the experimental error. The only major structural perturbation occurs near the novel chain termini in a surface loop of cpA16M-84, which becomes destabilized and rearranged. The results of this study are interpreted to show that: (1) several circular permutations in the compact jellyroll domain of the 1,3-1,4-beta-glucanases are tolerated without radical change of enzymatic activity or tertiary structure, (2) the three-dimensional structures of simple domains are encoded by the amino acid sequence with sufficient redundancy to tolerate a change in the sequential order of secondary structure elements along the sequence, and (3) the native N-terminal region is not needed to guide the folding polypeptide chain toward its native conformation.

Enzymology and folding of natural and engineered bacterial beta-glucanases studied by X-ray crystallography.

1,3-1,4-beta-D-glucan 4-glucanohydrolases (beta-glucanases) are synthesized in both plants and bacteria. The enzymes specifically hydrolyze beta-1,4 glycosyl bonds that are adjacent to beta-1,3 linkages in beta-glucan, a linear polysaccharide containing these bonds in an approximate ratio of 2.5:1. Here we review structural studies by X-ray crystallography of natural Bacillus beta-glucanases and engineered variants characterized by hybrid sequences, single-site mutations and circular permutations. In combination with biochemical data and site-directed mutagenesis, the crystallographic evidence permits the formulation of a likely reaction mechanism for the retaining Bacillus beta-glucanases. In addition, the shape of the active site channel, the known binding mode of a cellobioside epoxyalkyl inhibitor and the energy profile of the beta-glucan substrate explain the specificity of the enzymes for beta-glucan and the requirement for a beta-1,3 glycosyl bond next to the scissile bond. beta-Glucanases with circularly permuted sequences retain conformational stability, enzymatic activity and the native fold. The jellyroll tertiary structure of Bacillus beta-glucanases is remarkably stable, resisting changes in amino acid sequence, chain topology, ligand binding and crystal packing.