Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs.

An assessment of elastase-substrate kinetics and adsorption at the solid-liquid interface of peptide-bound resin was made in an approach to the solid-phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N-succinyl-alanine-alanine-proline-valine-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromogenic HNE substrate, was attached to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. To assess the enzyme-substrate reaction in a two-phase system, the kinetic profile of resin-bound peptide substrate hydrolysis by HNE was obtained. A glycine and di-glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate-resin and enzyme concentration. Enzyme hydrolysis of the resin-bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin-released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate-elastase adsorption and activity at the resin's solid-liquid interface for HNE detection with a solid-phase peptide.

Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution.

Dressings for chronic human wounds have been aimed at protection, removal of exudate, and improved appearance. However since the time of ancient Greece wound care and dressing strategies have primarily relied on empiricism. Recent studies have shown that chronic wounds contain high levels of tissue and cytokine destroying proteases including collagenase and neutrophil elastase. Therefore we sought to develop an effective wound dressing that could absorb elastase through affinity sequestration. Cotton gauze was modified by oxidation, phosphorylation, and sulfonation to enhance elastase affinity by ionic or active site uptake. Type VII absorbent cotton gauze was oxidized to dialdehyde cotton which was subsequently converted in part to the bisulfite addition product. Gauze preparations were also phosphorylated and carboxymethylated. Modified cotton gauzes were compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were soaked in oxidized, sulfonated, and phosphorylated cotton gauze showed reduced elastase activity. The initial velocities (v(o)) and turnover rates of elastase showed significant decreases compared with solutions taken from untreated gauze. The reduction in enzyme activity with dialdehyde cotton gauze was confirmed in solution by determining elastase inhibition with dialdehyde starch. The dialdehyde cotton gauze also decreased elastase activity in human wound fluid in a dose response relation based on weight of gauze per volume of wound fluid. Absorbency, pH, air permeability and strength properties of the modified gauze were also compared with untreated cotton gauze. This report shows the effect of reducing elastase activity in solution with cotton containing aldehydic or negatively charged cellulose fibers that may be applicable to treatment modalities in chronic wounds.

Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers.

The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells. Inhibition of B. subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme. Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis. Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed. The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129. The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics.

Synthesis and activity of NH2- and COOH-terminal elastase recognition sequences on cotton.

The application of peptide recognition sequences of elastase to fibers of wound dressings is a possible route to inhibiting high levels of destructive elastase in the chronic wound. For this reason we have synthesized the elastase recognition sequence Val-Pro-Val on both cotton cellulose, and carboxymethylated cellulose cotton (CMC) and prepared chromatography columns of these to examine elastase retention. The tripeptide was synthesized on cotton-based cellulose fibers both in sequence and as a tripeptide methyl ester. Glycine was employed as a linker of the recognition sequence to the cotton cellulose. Pre-treatment of cotton cellulose with cellulase improved the substitution level of glycine. The peptidocellulose conjugates were employed as a chromatographic stationary phase to assess elastase retention. The sequence Val-Pro-Val-OMe was amino-terminally anchored to carboxymethylated cotton and demonstrated retention of up to 58% of elastase when first applied to the column. Higher repetitive retention was demonstrated subsequently. Cotton gauze similarly modified with Val-Pro-Val-Gly cellulose was compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were treated with Val-Pro-Val-Gly cellulose cotton gauze, demonstrated reduced elastase activity. This study demonstrates the use of elastase recognition sequences as sequestering agents of elastase when attached to cotton fibers and constitutes a model for the design of peptidocellulose analogs in dressing fibers for chronic wounds.

Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on fibers and may be employed to evaluate structure vs. function of elastase inhibition in the modified fibers of wound dressing composites.

Heart rate and blood pressure resting levels and responses to generic stressors in Vietnam veterans with posttraumatic stress disorder.

Resting heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured on 3 successive mornings in the homes of drug-free Vietnam combat veterans, classified on the basis of DSM-III-R criteria into current posttraumatic stress disorder (PTSD; n = 20) or non-PTSD (n = 15). Responses to three generic stressor challenges (orthostatic, mental arithmetic, and cold pressor) were also measured. In the orthostatic stressor condition, DBP increased over time in the non-PTSD, but not in the PTSD, veterans, suggesting a paradoxically reduced autonomic response in PTSD. There were no other significant group differences in resting levels or responses to any of the challenges for any measure.

Omental transposition in chronic spinal cord injury.

The results of omental transposition in chronic spinal cord injury have been reported in 160 patients operated upon in the United States, Great Britain, China, Japan, India and Mexico, with detailed outcomes reported in few studies. Recovery of function to a greater degree than expected by natural history has been reported. In this series, 15 patients with chronic traumatic spinal cord injury (> 1.5 years from injury) underwent transposition of pedicled omentum to the area of the spinal cord injury. Of the first series of four patients who were operated upon in 1988, one died, one was lost to follow-up and two were followed with sequential neurological examinations and Magnetic Resonance Imaging (MRI) scans preoperatively, at 1 year post injury and 4 1/2 years post injury. Another 11 patients were operated in 1992 and underwent detailed neurological and neurophysiological examinations and had MRI scans preoperatively and every 4 months for at least 1 year after surgery. All patients completed a detailed self-report form. Of the total of 13 operated patients in both series followed for 1-4 1/2 years, six reported some enhanced function at 1 year and five of these felt the changes justified surgery primarily because of improved truncal control and decreased spasticity. MRI scans showed enlargement of the spinal cord as compared to preoperative scans in seven patients. Increased T2 signal intensity of the spinal cord was found by 1 year after surgery in eight of 13 operated patients. Neurophysiological examinations of 11 patients in the second series agreed with self-reports of increases or decreases in spasticity (r = 0.65, P < 0.03). Somatosensory evoked potentials and motor evoked potentials at 4 month intervals up to 1 year in these patients showed no change after surgery. Neurological testing, using the American Spinal Injury Association (ASIA) and International Medical Society of Paraplegia (IMSOP) international scoring standards, failed to show any significant changes when the 1-year post operative examination was compared to the first preoperative examination except for decreased sensory function after surgery which approached statistical significance. When the 11 patients in the second series were compared to eight non-operated matched patients, followed for a similar length of time, no significant differences were found. Complications encountered in the operated patients from both series included one postoperative death from a pulmonary embolus, one postoperative pneumonia, three chronic subcutaneous cerebrospinal fluid (CSF) fistulae requiring wound revision, and one patient who developed biceps and wrist extensor weakness bilaterally requiring graft removal. We conclude that the omental graft remains viable over time and this operation can induce anatomical changes in the spinal cord as judged by MRI. Some patients reported subjective improvement but this was not supported by objective testing. We, therefore, find no justification for further clinical trials of this procedure in patients who have complete or sensory incomplete lesions. Further testing in motor incomplete patients would seem appropriate only with compelling supportive data.

MDL-101,562 blocks the stimulatory effects of bombesin and gastrin-releasing peptide on hypothalamic dopaminergic neurons.

The effects of central administration of bombesin, gastrin-releasing peptide (GRP) and a putative bombesin/GRP receptor antagonist [Phe8 psi[CH2S]Leu9]litorin (MDL-101,562) were determined on the activities of hypothalamic tuberoinfundibular and periventricular-hypophysial dopaminergic neurons in male rats. Dopaminergic neuronal activity was estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence and the intermediate lobe of the posterior pituitary which contain terminals of these neurons. Intracerebroventricular injections of bombesin and GRP increased DOPAC concentrations in both the median eminence and intermediate lobe. MDL-101,562 had no effect on median eminence or intermediate lobe DOPAC concentrations per se, but blocked the stimulatory actions of bombesin and GRP on DOPAC concentrations in these regions. These results suggest that MDL-101,562 may be a useful pharmacologic tool in the study of the central actions of bombesin and related peptides.

Potent pseudopeptide bombesin-like agonists and antagonists. Correlation of ordered conformation of bombesin analogs to receptor activity.

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13-14 and 8-9, respectively, with psi[CH2S] and psi[CH2N(CH3)]. [Phe13 psi[CH2S]Leu14]bombesin and [Phe8 psi[CH2S]-Leu9]litorin bound to the murine pancreatic bombesin/gastrin releasing peptide receptor with similar dissociation constants (Kd = 3.9 and 3.4 nM, respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd = 1.6 nM and isomer II, Kd = 0.89 nM. Further oxidation to the sulfone decreased potency ([Phe8 psi[CH2SO2]Leu9]litorin, Kd = 9.9 nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a psi[CH2N(CH3)] at the 8-9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd = 0.1 nM) and [Leu9]litorin (Kd = 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa = 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the psi [CH2S]-containing antagonist.

Micelle-bound conformations of a bombesin/gastrin releasing peptide receptor agonist and an antagonist by two-dimensional NMR and restrained molecular dynamics.

Two nonapeptide analogs of the carboxyl termini of bombesin (Bn) and gastrin releasing peptide (GRP) have been synthesized. Despite the small difference in chemical composition between these peptides, one was a potent agonist and the other a potent antagonist of the Bn/GRP receptor in murine pancreas. All protons of both peptides, in dodecylphosphocholine micelles, were assigned by two-dimensional nuclear magnetic resonance spectroscopy. Interproton distance were derived from cross-peak volumes in nuclear Overhauser enhancement spectra. Conformations of both peptides were derived by distance-restrained molecular dynamics simulations using the interproton distances as constrains. The agonist conformation resembled a relaxed helix formed by three connected turns. The two N-terminal turns were similar for both peptides. The third turn of the agonist, at the carboxyl terminus, was absent in the antagonist. One interproton distance at the carboxyl terminus of the antagonist indicates that the chemical group connecting the last two residues of this peptide mimics a cis peptide bond geometry.

Isolation and liquid chromatographic determination of the cyclic peptide mycotoxin cyclosporin A from rice.

A simple method for determination and quantitation of a cyclic peptide mycotoxin, cyclosporin A, in rice is presented. Rice inoculated with Trichoderma polysporum (Link ex Pers.) was extracted with methylene chloride after 4 weeks of incubation. Cyclosporin A was isolated from extracts by using open bed gel filtration column chromatography (LH-20, acetonitrile) and monitored with thin layer chromatography and reverse phase liquid chromatography coelution with a standard. Preliminary thin layer chromatographic methods were developed. Cyclosporin A was detected by iodine and after partial acid hydrolysis by ninhydrin and UV light. A liquid chromatographic method was developed that used a reverse phase disposable cartridge cleanup and isocratic chromatography with a reverse phase octadecylsilica column and a UV detector set at 212 nm. Recovery of cyclosporin A from spiked rice samples (mg/g range) was 85%.

In vitro activity profiles of cyclic and linear enkephalin pseudopeptide analogs.

The peptide bond in the 4-5 position of the cyclic and linear enkephalin analogs H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] and H-Tyr-D-Ala-Gly-Phe-L(or D)-Leu-OH was replaced by a thiomethylene ether linkage. Each of the configurational isomers of the cyclic pseudopeptide H-Tyr-cyclo[-D-Lys-Gly-Phe psi [CH2S]L(or D)-Leu-] showed high potency in both the guinea pig ileum and the mouse vas deferens assay and, therefore, had no preference for either mu- or delta-opioid receptors, in contrast to the cyclic parent peptides H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] which are mu-receptor selective. The loss of selectivity observed with the cyclic pseudopeptides may be due to the greater flexibility of their 18-membered ring structures as a consequence of the peptide bond substitution. The linear pseudopeptide analogs were both less potent and less delta-receptor selective than their parent compounds. These results indicate that thiomethylene ether peptide bond replacements can have a pronounced effect on the activity profile of peptide hormones and neurotransmitters.

Structure-activity relationships of enkephalins containing serially replaced thiomethylene amide bond surrogates.

An isomeric series of four leucine-enkephalin analogs containing the thiomethylene ether unit as an amide bond replacement in all positions have been prepared by solid phase methods. The resulting pseudopeptides divulged widely differing retentive behaviors on reversed phase high performance liquid chromatography (HPLC). An analog containing the Phe psi[CH2S]Leu dipeptide replacement at the 4-5 position exhibited binding close to the parent, leucine enkephalin; its guinea pig ileum (GPI) activity was the highest of the analogs tested. Another compound, Tyr psi[CH2S]Gly1-2]-Leu-enkephalin, also displaced 3H-etorphine well in the binding assay, but caused increased contractions in the GPI assay at low concentrations. The Phe psi[CH2S]Leu results are not compatible with the necessity of a beta-turn structure for agonist activity in the GPI assay.