Incidence of contralateral occult inguinal hernia found at the time of laparoscopic trans-abdominal pre-peritoneal (TAPP) repair.

BACKGROUND: Trans-abdominal laparoscopic inguinal hernia repair allows rapid assessment and exploration of the contralateral groin and repair of an occult hernia. Although previous studies have shown that the totally extra-peritoneal (TEP) hernia repair can be used to assess the contralateral groin, there is little data pertaining to the trans-abdominal pre-peritoneal (TAPP) approach. The aim of this study was to document the incidence of occult contralateral hernia at the time of TAPP hernia repair. METHODS: Data were collected prospectively from all patients undergoing laparoscopic TAPP hernia repair in a District General Hospital over a three-year period. Two specialist laparoscopic/upper gastrointestinal surgeons undertook all of the operations and telephone follow-up was carried out by a dedicated laparoscopic specialist nurse. RESULTS: A total of 310 patients underwent hernia surgery. Four cases were excluded, leaving 306 patients in the study. The male:female ratio was 10.5:1, with a median age of 59 years. Two hundred and six (67%) patients were booked for a unilateral hernia repair; of these, a contralateral hernia was found and repaired in 45 (22%). In 76 cases where a bilateral repair was planned, 61 (80%) went on to have both groin defects repaired. In the remaining 20%, the clinical suspicion of bilateral hernia was revised at the time of surgery to unilateral only. Twenty (7%) patients were booked to undergo a unilateral repair with the possibility of a contralateral hernia--in this group, the suspected contralateral defect was confirmed in 6 (30%) cases. Four (1%) cases were booked as femoral repairs, one of which was found to be an inguinal hernia. The clinical diagnostic accuracy was 78%. CONCLUSION: Accurate incidence figures of an occult contralateral inguinal hernia will enhance the pre-operative information given to patients and may impact on resource allocation and planning theatre logistics. Finding and repairing an occult contralateral hernia at the time of TAPP has the distinct advantage that it saves the patient from further symptoms and from another operation with its associated potential morbidity.

Structure-function analysis of a phage display-derived peptide that binds to insulin-like growth factor binding protein 1.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family.

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

Peptide exosite inhibitors of factor VIIa as anticoagulants.

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.

Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment.

Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.

Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.

Molecular mimics of insulin-like growth factor 1 (IGF-1) for inhibiting IGF-1: IGF-binding protein interactions.

IGF-1 (insulin-like growth factor 1) is a 70-residue protein hormone which has both metabolic and mitogenic activities mediated through IGF-1 binding to cell surface receptors. However, an unrelated class of proteins, the IGF-binding proteins (IGFBPs) also bind IGF-1 in the serum and tissues and block or modulate its activity in vivo. Therefore, inhibitors of the IGFBPs can alter the distribution between free and bound IGF-1 [Loddick, S. A., Liu, X.-J., Lu, Z.-X., Liu, C., Behan, D. P., Chalmers, D. C., Foster, A. C., Vale, W. W., Ling, N., and De Souza, E. B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898] and potentially affect the distribution of IGF-1 among body tissues. We report here that phage-displayed peptide libraries have yielded a peptide that binds IGFBP-1 and produces IGF-like activity at sub-micromolar concentrations. The 14-residue peptide has an extremely well-defined solution conformation that can aid in the design of smaller, orally active compounds. Interestingly, the peptide structure contains a helix, as does one region of IGF-1 previously implicated in IGFBP binding, yet displays side chains different from those of the IGF-1 helix I. Furthermore, an IGF-1 variant lacking receptor-signaling activity in vitro is shown here to produce IGF-like mitogenic and metabolic activity in vivo. These results suggest that small antagonist mimetics of protein ligands, identified by binding selection to otherwise inhibitory factors, may be useful as indirect agonists for a variety of therapeutic applications.

Model peptide studies demonstrate that amphipathic secondary structures can be recognized by the chaperonin GroEL (cpn60).

The molecular chaperone cpn60 binds many unfolded proteins and facilitates their proper folding. Synthetic peptides have been used to probe the question of how cpn60 might recognize such a diverse set of unfolded proteins. Three hybrid peptides were synthesized encompassing portions of the bee venom peptide, apamin, and the sequence KWLAESVRAGK from an amphipathic helix in the NH2-terminal region of bovine rhodanese. Two disulfides connecting cysteine residues hold the peptides in stable helical conformations with unobstructed faces oriented away from the disulfides. Peptides were designed to present either a hydrophobic or hydrophilic face of the amphipathic helix that is similar to the one near the amino terminus of rhodanese. Aggregation of these peptides was detected by measuring 1,1'-bis(4-anilino)napthalene-5,5'-disulfonic acid (bisANS) fluorescence at increasing peptide concentrations, and aggregation was not apparent below 2 microM. Thus, all experiments with the peptides were performed at a concentration of 1 microM. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a Kd = approximately 106 microM for binding to cpn60, whereas there was no detectable binding of the reduced form. The peptide with the exposed hydrophilic face did not bind to cpn60 in either the oxidized or reduced states. Fluorescence experiments utilizing bisANS as a probe showed that binding of the helical hydrophobic peptide could induce the exposure of hydrophobic surfaces on cpn60, whereas the same peptide in its random coil form had no effect. Thus, binding to cpn60 is favored by a secondary structure that organizes and exposes a hydrophobic surface, a feature found in amphipathic helices. Further, the binding of a hydrophobic surface to cpn60 can induce further exposure of complementary surfaces on cpn60 complexes, thus amplifying interactions available for target proteins.

RhNGF slow unfolding is not due to proline isomerization: possibility of a cystine knot loop-threading mechanism.

The unfolding of recombinant human beta-NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 degrees C. Fluorescence spectroscopy, size-exclusion and reversed-phase HPLC, ultra-centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little beta-sheet structure, but retained residual structure in the tryptophan indole and high-field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half-life of 23 h at 25 degrees C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N-terminus. A model for the slow unfolding reaction is proposed where the N-terminus threads through the cystine knot to form M2, a loop-threading reaction, increasing the conformational freedom of the denatured state.

High-resolution solution structure of the EGF-like domain of heregulin-alpha.

The solution structure of the 63-residue heregulin-alpha (HRG-alpha) epidermal growth factor (EGF)-like domain, corresponding to residues 177-239 of HRG-alpha, has been determined to high resolution using data from two-dimensional and three-dimensional homo- and heteronuclear NMR spectroscopy. The structure is based on a total of 887 internuclear distance and dihedral restraints derived from data obtained using unlabeled and uniformly 15N-labeled protein samples, at pH 4.5, 20 degrees C. A total of 20 structures were calculated using a hybrid distance geometry-simulated annealing approach with the program DGII, followed by restrained molecular dynamics using the program DISCOVER. The average maximum violations are 0.12 +/- 0.01 angstroms and 1.4 +/- 0.3 degrees for distance and dihedral restraints, respectively. The backbone (N,C(alpha),C) atomic rms distribution about the mean coordinates for residues 3-23 and 31-49 is 0.29 +/- 0/07 angstroms. The N-and C-terminal residues (1-2 and 50-63) and 24-30 are disordered. Comparison of the HRG-alpha EGF-like domain structure with the previously determined structure of human EGF [Hommel et al. (1992) J. Mol. Biol. 227, 271-282] reveals a high degree of structural similarity; excluding the N-terminal region (residues 1-13), the disordered phi-loop region (residues 24-30) that contains a three-residue insertion in HRG-alpha relative to hEGF, and the disordered C-terminal region (residues 50-63), the C(alpha) alignment between the HRG-alpha and hEGF minimized mean structures has a rms difference of approximately 1 angstrom. In HRG-alpha the N-terminal residues 2-6 form a well-defined beta strand rather than being disordered as found for hEGF. This structural difference correlates with functional data which suggest that the N-terminal region of the HRG-alpha EGF-like domain is responsible for the observed receptor specificity differences between HRG-alpha and EGF.

Proton NMR assignments and solution conformation of RANTES, a chemokine of the C-C type.

1H NMR has been used to investigate the structural properties of RANTES, a protein from the C-C branch of the chemotactic cytokine family that has a strong chemoattractive effect on monocytes, lymphocytes, and eosinophils. Titration of pH from 5.0 to 2.5 indicates that RANTES is extensively aggregated in solution above pH 4.0. At pH 3.7 the protein is mostly dimeric, although this species does dissociate to the monomer with a Kd of 35 microM. NMR data have been acquired and resonance assignments made for the dimeric species. Structures of the dimer have been generated by distance geometry and simulated annealing calculations that utilized 1956 intramolecular distance restraints, 120 intermolecular distance restraints, 164 dihedral angle restraints, and 68 restraints enforcing 34 hydrogen bonds (17.0 restraints per residue). The structure is well-defined (average root mean square deviation from the average structure of 0.38 +/- 0.06 and 0.53 +/- 0.12 A for backbone heavy atoms of residues 4-66 of the monomer and dimer, respectively). Each monomer consists of a C-terminal alpha-helix packing against a three-stranded antiparallel beta-sheet and two short N-terminal beta-strands; dimerization occurs between the N-terminal regions of each monomer. This quaternary structure is very different from that of the C-X-C chemokines such as interleukin-8 and melanoma growth stimulatory activity but similar to that found for the C-C chemokine macrophage inflammatory factor 1 beta. Distinct structural differences between RANTES and other chemokines at both the tertiary and quaternary level are discussed with regard to the distinct biological functions of the C-C and C-X-C members of this protein family.

Determination of the solution structure of the peptide hormone guanylin: observation of a novel form of topological stereoisomerism.

Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds. Guanylin shares sequence similarity with the bacterial heat-stable enterotoxin (STa) and is capable of binding to and stimulating the STa guanylyl cyclase receptor. Biologically active peptides have been prepared by two methods: (1) enzymatic treatment of a 99 residue proprotein (denoted proguanylin) expressed in Escherichia coli and (2) solid-phase chemical synthesis. Although both sources yield material that is pure by high-performance liquid chromatography and mass spectrometry, analysis by nuclear magnetic resonance (NMR) indicates that peptides from both sources contain two conformationally distinct species present in a 1:1 ratio. The chemical shift differences between the two species are large, allowing unambiguous sequential NMR assignments to be made for both sets of resonances. Exchange between the two forms was not observed even at 70 degrees C. Structural restraints have been generated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometry and restrained energy minimization. The resulting structures for the first isoform are well defined (root-mean-square deviation from the average structure for backbone atoms of 0.47 A) and adopt a right-handed spiral conformation, similar to that observed for heat stable enterotoxin. The second isoform is less well defined (root-mean-square deviation from the average structure for backbone atoms of 1.07 A) but clearly adopts a very different fold consisting of a left-hand spiral. The differences in structure suggest that the two forms may have very different affinities toward the STa receptor. The observation of such isomerism has important implications for the common practice of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity.

Case report: frequent migraines with aura in a patient with a germ cell tumor.

Accelerated migraine with aura has not been previously described in association with germ cell tumor. The following case describes a likely rare paraneoplastic syndrome in a patient with a germ cell tumor with seminomatous elements.