Fasciotomy for chronic exertional compartment syndrome of the leg: clinical outcome in a large retrospective cohort.

BACKGROUND: Chronic exertional compartment syndrome (CECS) is an overuse disorder typically affecting an athletic population. CECS is a diagnosis based on history and intracompartmental pressure (ICP) testing. CECS patients can be treated surgically by fasciotomy; however, research on the relationship between ICP and patient symptoms and also between ICP and patient-reported outcome post-fasciotomy is limited. This study aims to (1) assess functional outcome and patient satisfaction post-fasciotomy and (2) identify any potential correlation between ICP and reported levels of pain. METHODS: 138 CECS patients who had ICP measurements and subsequently underwent fasciotomy were identified from our regional service for exercise-induced lower limb extremity pain between January 2000 and March 2017. Clinical outcomes were recorded at the time of ICP testing and in the post-operative follow-up clinic. Pain was reported using a verbal rating scale (VRS) ('low', 'moderate' or 'high') or as a visual analogue score (VAS) 0-10 (0 = least painful, 10 = most painful). Spearman's ranked correlation test was used to calculate correlation between ICP and reported pain. RESULTS: A total of 138 patients were eligible for inclusion in this study (mean age 29.7 ± 9.7 years, 110 M, 28 F) of which 109 patients (VRS n = 61, VAS n = 48) reported pain level at pre- and post-operative stages. Mean pre-operative VAS score was 8.52 ± 0.71, and decreased to 0.77 ± 0.69 post-operatively. An insignificant positive correlation (r = 0.046, two-tailed p = 0.76) was found between VAS pain and ICP. A significant moderate positive correlation (r = 0.497, two-tailed p = 0.01) was found between VRS pain and ICP. CONCLUSION: Fasciotomy significantly reduces pain and increases activity levels in CECS patients. ICP was found to positively correlate with patient-reported pain.

Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells.

Coronavirus envelope (E) protein is a small integral membrane protein with multi-functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N- and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N-terminus exposed luminally and the C-terminus exposed cytoplasmically (N(exo(lum)-)C(cyto)). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV.

Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein.

A diverse group of cytolytic animal viruses encodes small, hydrophobic proteins to modify host cell membrane permeability to ions and small molecules during their infection cycles. In this study, we show that expression of the SARS-CoV E protein in mammalian cells alters the membrane permeability of these cells. Immunofluorescent staining and cell fractionation studies demonstrate that this protein is an integral membrane protein. It is mainly localized to the ER and the Golgi apparatus. The protein can be translocated to the cell surface and is partially associated with lipid rafts. Further biochemical characterization of the protein reveals that it is posttranslationally modified by palmitoylation on all three cysteine residues. Systematic mutagenesis studies confirm that the membrane permeabilizing activity of the SARS-CoV E protein is associated with its transmembrane domain.

Biochemical and functional characterization of Epstein-Barr virus-encoded BARF1 protein: interaction with human hTid1 protein facilitates its maturation and secretion.

EBV BARF1 gene encodes a secretory protein with transforming and mitogenic activities. In this report, the post-translational modification, folding, maturation and secretion of BARF1 are systematically studied by site-directed mutagenesis and overexpression of the protein in mammalian cells using the vaccinia/T7 system. The protein was shown to be post-translationally modified by N-linked glycosylation on the asparagine 95 residue. This modification was confirmed to be essential for the maturation and secretion of the protein. Analysis of the four cysteine residues by site-directed mutagenesis demonstrated that cysteine 146 and 201 were essential for proper folding and secretion of the protein. To search for human proteins involved in the maturation process of the protein, a yeast two-hybrid screening was carried out using the BARF1 sequence from amino acids 21-221 (BARF1Delta) as bait, leading to the identification of human hTid1 protein as a potential interacting protein. This interaction was subsequently confirmed by coimmunoprecipitation and dual immunofluorescent labeling of cells coexpressing BARF1 and hTid1, and the interaction domain in hTid1 was mapped to amino acids 149-320. Interestingly, coexpression of BARF1 with hTid1 demonstrated that hTid1 could promote secretion of BARF1, suggesting that hTid1 may act as a chaperone to facilitate the folding, processing and maturation of BARF1.

Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability.

To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in Escherichia coli. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in E. coli cells.

Ninhydrin as a reversible protecting group of amino-terminal cysteine.

The proximity of the alpha-amine and beta-thiol of alpha-amino terminal-cysteine (NT-Cys) residues in peptides imparts unique chemical properties that have been exploited for inter- and intra-molecular ligation of unprotected peptides obtained through both synthetic and biological means. A reversible protecting group orthogonal to other protection strategies and reversible under mild conditions would be useful in simplifying the synthesis, cleavage, purification and handling of such NT-Cys peptides. It could also be useful for the sequential ligation of peptides. To this end, we explored tri-one chemistry and found that ninhydrin (indane-1,2,3 trione) reacted readily with cysteine or an NT-Cys-containing peptide on- or off-resin at pH 2-5 to form Ninhydrin-protected Cys (Nin-Cys) as a thiazolidine (Thz). The Thz ring, protecting both the amino and thiol groups in Nin-Cys, completely avoids the formylation and Thz side reactions found during hydrofluoric acid (HF) cleavage when N-pi-benzyloxymethyl histidine groups are present. Nin-Cys is stable during coupling reactions and various cleavage conditions with trifluoroacetic acid or HF, but is deprotected under thiolytic or reducing conditions. These properties enable a facile one-step deprotection and end-to-end-cyclization reaction of Nin-Cys peptides containing C-terminal thioesters.

Tandem ligation of unprotected peptides through thiaprolyl and cysteinyl bonds in water.

Tandem ligation for the synthesis and modification of proteins entails forming two or more regiospecific amide bonds of multiple free peptide segments without a protecting-group scheme. We here describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the N --> C direction, first by an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both an NT-Cys and a CT-thioester. The newly created O-ester-linked segment with a CT-thioester was then ligated to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently, this basic condition also catalyzed the O,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides ranging from 19 to 70 amino acids, including thiaproline --> proline analogues of somatostatins and two CC-chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for combinatorial segment synthesis.

A thioester ligation approach to amphipathic bicyclic peptide library.

An efficient approach to synthesize an amphipathic bicyclic peptide library from unprotected peptides is demonstrated through an on-resin intramolecular thioester ligation and an off-resin DMSO-mediated disulfide formation.

Design of Gram-negative selective antimicrobial peptides.

Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterial invasion and triggers defensive host responses. However, excessive responses also lead to the serious pathophysiological consequence of septic shock. To develop Gram-negative selective compounds that can inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptides based on a cystine-stabilized beta-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eight amino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracing disulfides. NMR study of K4A, an R4A analogue with four Arg --> Lys replacements, confirmed the amphipathic design elements with four Lys on one face of the antiparallel beta-strand and two hydrophobic cystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potency and Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala and Gly with Arg or bulky hydrophobic amino acids displayed increased potency and selectivity in both low- and high-salt conditions. Analogues R5L and R6Y containing additional cationic and bulky hydrophobic amino acids proved the best mimics of the amphipathic topology of the "active-site" beta-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitory concentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggest that an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.

Tandem peptide ligation for synthetic and natural biologicals.

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.

Subtilisin-catalyzed synthesis of amino acid and peptide esters. Application in a two-step enzymatic ligation strategy.

We describe an efficient enzymatic approach to the synthesis of amino acid and peptide esters. The serine protease subtilisin Carlsberg (EC 3.4.21.62) was found to efficiently catalyze the specific formation of C(alpha)-carboxyl 3-hydroxypropyl or 4-hydroxybutyl esters of certain Boc-amino acids and peptides in high-content 1,3-propanediol or 1,4-butanediol solution, with substrate specificity parallel to that of the normal hydrolytic reaction. This approach can be coupled with kinetic-control reverse proteolysis in a two-step enzymatic peptide ligation scheme. [reaction: see text]

Production of antipeptide antisera.

This unit describes methods used for the chemical coupling of synthetic peptides to carrier proteins, required for the preparation of peptide immunogens. The carrier protein described here is keyhole limpet hemocyanin (KLH) because it is the one most commonly used. However, other proteins may be used in place of KLH, including bovine serum albumin (BSA) and ovalbumin. Coupling may be accomplished as described with MBS, which requires a Cys residue in the peptide, or with glutaraldehyde, EDCI, or BDB. Also included are an assay for detecting free sulfhydryl (SH) groups, a means of calculating coupling efficiency, an immunization schedule, an indirect ELISA, and a method for preparing a peptide affinity column. The final methodology described is the multiple antigen (MAP) system.

Synthesis and application of peptide dendrimers as protein mimetics.

The use of peptides to mimic a portion of a protein structure is a challenging and powerful tool in the discovery of new drugs. In native proteins, discontinuous bioactive peptide surfaces are held together in a particular conformation by the structural rigidity of the protein. Approaches to mimicking a structural surface center on bringing the potential peptide sequences together by assembling the peptide chains on a template. These templates can be flexible dendrimeric or cyclic peptides as well as more rigid organic molecules. The Multiple Antigen Peptide (MAP) system represents a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. A variation of this procedure, the cyclic Multiple Antigen Peptide (cMAP) approach, is also presented here. Having branched multiple closed-chain architectures, the cMAP system is often a superior approach for protein mimetics because the multiple constrained peptides can mimic bioactive conformations. Whether to select this approach over MAP depends on the properties of the peptides, but usually if the peptides are too small to adopt a stable conformation on their own, incorporation of a cyclic structure may be necessary. MAPs have been applied to areas of study such as inhibitors, artificial proteins, affinity purifications, and intracellular transport.

Methods and strategies of peptide ligation.

This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.

Dehydropeptides from orthogonal ligation of unprotected peptides.

A facile method has been developed to synthesize linear and cyclic dehydropeptides from unprotected peptide precursors. This method exploits an N-terminal Cys for a Cys-thioester ligation to generate an unprotected peptide and as a precursor for conversion to DeltaAla by beta-elimination under mild conditions.

Solid-phase synthesis of 1,2,3, 4-tetrahydro-beta-carboline-containing peptidomimetics.

A solid-phase method for the synthesis of 1,2,3, 4-tetrahydro-beta-carboline-containing peptidomimetics has been developed. The key step in the strategy is the Pictet-Spengler condensation of a resin-bound tryptophan-containing fragment with an Fmoc-amino aldehyde.

Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures.

Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.

Membranolytic selectivity of cystine-stabilized cyclic protegrins.

To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin-1 (PG-1), an 18-residue cationic peptide with a broad-spectrum antimicrobial activity. These cyclic protegrins bear end-to-end peptide bonds together with varying numbers (zero to three) of cross-strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low- and high-salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram-positive and Gram-negative bacteria, fungi and one tested virus, HIV-1. Compared to PG-1, the cyclic tricystine ccPG 3 displayed approximately a 10-fold decrease in hemolytic activity against human cells and 6- to 30-fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [DeltaSS]cPG 8, a cyclic protegrin with no disulfide bond, and [DeltaCys6,15]cPG 5, a cyclic mimic of PG-1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG-1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of beta-strand structures in water/trifluoroethanol or phosphate-buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.

Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells.

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.

Marked increase in membranolytic selectivity of novel cyclic tachyplesins constrained with an antiparallel two-beta strand cystine knot framework.

We have developed a highly constrained 18-residue cyclic peptide template based on the antimicrobial peptide tachyplesin-1 that features an end-to-end peptide backbone and a cystine knot-like motif with three evenly spaced disulfide bonds to cross-brace the antiparallel beta-strands and to approximate an amphiphatic "beta-tile"-like structure. Six beta-tile analogs were prepared to correlate different topological patterns with membranolytic specificity. Their conformations and antimicrobial and hemolytic activities were compared with tachyplesin-1 and the recently discovered Rhesus monkey theta defensin (RTD) which contains similar beta-tile structural elements. The beta-tile peptides and RTD retained broad spectrum antimicrobial activities. In general, they were less active than tachyplesin-1 in 10 tested organisms but their activity increased under high-salt (100 mM NaCl) rather than in low-salt conditions. The beta-tile peptides are highly nontoxic to human erythrocytes with EC(25) ranging from 600 to 4000 microM. Collectively, our results show that the design of a highly rigid peptide template is useful for further analog study to dissociate antimicrobial activity from cytotoxicity which would be helpful in discovering clinical applications for peptide antibiotics.