Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour.

OBJECTIVES: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour. METHODS: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. RESULTS: fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca2+]i. CONCLUSIONS: These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori.

Helicobacter pylori is believed to trigger neutrophil activation through several factors, including the H. pylori neutrophil-activating protein (HpNAP). The aim of this study was to characterise the factors within H. pylori cell-free extracts that stimulate neutrophil activation. Neutrophil activation was found to be dose-dependent and exhibited considerable variation between different clinical isolates. Activity was attributable to more than one protein factor. A low mol. wt fraction of <3 kDa was found to contribute a large proportion of the neutrophil-stimulating activity within H. pylori cell-free extract. Additional activity was provided by a high mol. wt fraction, possibly representing HpNAP. An inhibition ELISA and neutralisation experiments failed to identify or exclude formyl methionyl leucyl phenylalanine as the active factor within the low mol. wt fraction. The importance of the putative, low mol. wt neutrophil-activating factor may have been overlooked by those studies that have used concentrated H. pylori extracts.

Pharmacologic properties of brewery dust extracts in vitro.

STUDY OBJECTIVES: To study the effects of extracts of brewery dust on isolated guinea pig tracheal smooth muscle in vitro. DESIGN: Parallel pharmacologic intervention on guinea pig tracheal rings that were obtained from the same animal. SETTING: Mount Sinai School of Medicine, Department of Pulmonary Medicine. MATERIAL: The isolated guinea pig tracheal tissue of 18 guinea pigs. INTERVENTIONS: Pretreatment of guinea pig rings by mediator-modifying agents before challenge with the brewery dust extracts. MEASUREMENTS AND RESULTS: The effect of brewery dust extracts on isolated guinea pig tracheal smooth muscle was studied using water-soluble extracts of dust obtained from brewery materials, including hops, barley, and brewery yeast. Dust extracts were prepared as a 1:10 (wt/vol) aqueous solution. Dose-related contractions of nonsensitized guinea pig tracheas were demonstrated using these extracts. The dust extracts contained significant quantities of bacterial components (eg, endotoxin and n-formyl-methionyl-leucyl-phenylalanine), but these agents were not thought to contribute directly to the constrictor effect of the dusts. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with the following drugs known to modulate smooth muscle contraction: atropine; indomethacin; pyrilamine; LY171883; nordihydroguaiaretic acid; captopril; thiorphan; verapamil; and TMB8. The constrictor effects of the dust extracts were inhibited by a wide variety of agents, the patterns of which depended on the dust extract. Atropine consistently and strikingly reduced the contractile effects of these extracts. These observations may suggest an interaction of the extracts with parasympathetic nerves or, more directly, with muscarinic receptors. The inhibition of contraction by the blocking of other mediators was less effective and varied with the dust extract. CONCLUSIONS: We suggest that brewery dust extracts cause a dose-related airway smooth muscle constriction by nonimmunologic mechanisms involving a variety of airway mediators and, possibly, cholinergic receptors. This effect is not dependent on presensitization of the guinea pigs.

Measurement of environmental formylmethionyl-peptides.

Formylmethionyl-peptides are naturally occurring, biologically active ligands produced by bacteria. They produce a variety of biological effects including neutrophil chemotaxis, cellular degranulation, oxygen-free radical production, and smooth muscle contraction. Our studies have demonstrated that oxidized and reduced forms of formylmethionyl-leucyl-phenylalanine (fMLP) can be detected in bulk environmental organic dust samples. Organic dust fMLP content may not reflect total formylmethionyl-peptide content and pathological sequelae. Attempts to develop a total formylmethionyl-peptide assay that would reflect its pathological potential have thus far been unsuccessful. Information has been derived concerning the biology of formylmethionyl-peptides from these studies. Chromatographic, radioenzymatic, and radioreceptor-ligand binding studies were performed. High-performance liquid chromatography (HPLC) analysis of synthetic and environmental fMLP demonstrated that fMLP is labile, forming three oxidation products. HPLC is limited by inadequate sensitivity for air sample analysis and the probability of the presence of multiple formylmethionyl-peptides. Deformylases were isolated from Escherichia coli, but their usefulness in a competitive assay to detect formylmethionyl-peptides was limited by specificity differences from that for biological receptors. Receptor binding studies were conducted in an attempt to replace the deformylase with a biological receptor. The receptor binding patterns noted were consistent with the existence of three distinct formylmethionyl-peptide receptor subsets in neutrophils and alveolar macrophages. The plurality of fMLP receptor subtypes interfered with formylmethionyl-peptide measurement in a competitive assay. Formylmethionyl-peptides may contribute to organic dust-induced disease, but better techniques for the assessment of exposure to these agents are needed to properly assess their health impact.

[The influence of vitamin A on production of oxygen free radicals and activity of granulocyte catalase in patients with chronic bronchitis]. / Wplyw witaminy A na wytwarzanie wolnych rodników tlenowych i aktywnosc katalazy granulocytów u chorych na przewlekle zapalenie oskrzeli.

The effect of vitamin A on granulocyte chemiluminescence (CT) and catalase activity was studied in 16 patients with COPD. The results obtained show a significant decrease of CL and a significant increase of granulocyte CT activity after incubation with vitamin A, particularly in nonsmoking subjects. This study showed a significant decrease of CT activity after incubation of granulocyte with hydralazine both in cigarette smokers or nonsmokers.

Capillary zone electrophoresis-mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Mixtures of peptides have been analyzed by capillary zone electrophoresis in conjunction with mass spectrometry (MS) using an on-line coaxial continuous-flow fast atom bombardment interface. MS and MS-MS spectra have been acquired in electrophoretic real time from femtomole levels of the peptides, while maintaining separation efficiencies in excess of 100,000 theoretical plates.

Radio-immunoassay for formyl methionyl leucyl phenylalanine. I. Development and application to assessment of chemotactic peptide production by enteric bacteria.

Bacterial chemotactic peptides are low molecular weight peptides which stimulate a wide range of neutrophil functions following binding to specific leucocyte receptors. Formyl methionyl leucyl phenylalanine (FMLP) is the major chemotactic peptide in Escherichia coli culture supernatants. This paper reports the development and validation of a radio-immunoassay (RIA) for FMLP and its application to the analysis of formyl peptide production by enteric bacteria in vitro. The assay was moderately sensitive (10 nmol/L FMLP) and highly specific showing cross reactivity with F-met-leu-tyr, F-nle-leu-phe and F-met-met-met sequences (ID50 = 200, 100 and 250 nmol/L, respectively) but no significant cross reactivity with non-formylated or other formylated di- and tri-peptides (ID50 = 10(5) nmol/L. Culture supernatants from five species of enteric bacteria were filtered, concentrated and fractionated by reverse phase high performance liquid chromatography before RIA. All five organisms produced immunoreactive F-met peptides. A major peak of immunoreactivity co-chromatographing with authentic FMLP was found in all supernatants, but additional peaks representing more hydrophobic peptides were found in Streptococcus faecalis and Bacteroides fragilis cultures. In E. coli culture supernatants, concentration of immunoreactive FMLP increased in a linear fashion during 3 h of log phase growth reaching 31.2 nmol/L(s.e.m. = 10) with final bacterial concentrations of 3 +/- 0.73 x 10(8)/mL (n = 6). These findings extend earlier work showing production of bioactive formyl oligopeptides by different species of enteric bacteria and suggest that a RIA for FMLP will be a useful tool for investigating the production and metabolic fate of such peptides in man.

Radio-immunoassay for formyl methionyl leucyl phenylalanine. II. Demonstration of an enterohepatic circulation of immunoreactive bacterial chemotactic peptides in man.

Bacterial chemotactic peptides (F-met-oligopeptides) are secreted by several species of commensal enteric bacteria and can be assayed by bioassay techniques in human colonic luminal fluid. We have previously demonstrated intestinal absorption and enterohepatic circulation of radiolabelled F-met peptides introduced into rat colon, and an eightfold increase in absorption and biliary excretion in rats with experimental colitis. This paper describes the application of a radio-immunoassay to measurements of formyl oligopeptides in human faecal dialysates, colonic and systemic venous blood and bile. All samples were fractionated by reverse-phase high performance liquid chromatography (HPLC) prior to assay. Immunoreactivity was found in faecal dialysates (5-700 nmol/L F-met-leu-phe equivalents) and bile samples (3-150 nmol/L) from normal subjects. After HPLC fractionation, up to five distinct peaks of immunoreactivity were identified. One of these co-chromatographed with authentic F-met-leu-phe; the others probably represented either closely related peptides or peptides of different chain lengths originating from the same F-met-leu-phe precursor protein. Colonic venous blood from two patients with ulcerative colitis contained immunoreactive peptide (10-30 nmol/L) and substantial immunoreactivity was found in ileostomy fluid and bile from two patients with primary sclerosing cholangitis. These results suggest the presence of an enterohepatic circulation of bacterial F-met oligopeptides in man and provide a basis for studies of the role of such pro-inflammatory peptides in patients with inflammatory bowel disease and associated hepatobiliary disorders.

Conformational energy analysis of the chemotactic tripeptide formyl-Met-Leu-Phe and three analogs.

Conformational energy analyses were carried out on the chemotactic tripeptide fMLF (CHO-Met-Leu-Phe) and three analogs fALF (CHO-Ala-Leu-Phe). fMF (CHO-Met-Phe), and MLF (H-Met-Leu-Phe). A near-folded or puckered conformation predominates in all four peptides. The calculated average end-to-end distance R of each of the peptides is 7.4 A, 7.6 A, 7.0 A, and 7.3 A, respectively (where bends have R less than or equal to 7 A and extended structures have R approximately 10.5 A). The puckered conformation calculated for fMLF is similar to that determined experimentally for fMLF in nonpolar solvents and in the protein receptor. The results suggest that maximum chemotactic activity of the peptides depends on a combination of the chemical structure (the presence of N-formyl-methionine) and backbone conformation (C7conformation of the first amino acid residue).

The state of the N-terminus of recombinant proteins: determination of N-terminal methionine (formylated, acetylated, or free).

The removal of N-terminal methionine from proteins produced by recombinant DNA techniques is often far from quantitative. Furthermore, a proportion of the methionylated product may be N alpha-blocked and thus not easily accessible to conventional (Edman) techniques of protein characterization. In this paper, a method for overcoming the resulting analytical problems is described. The technique is based on perdeuteroacetylation (performed only if unblocked methionine is to be determined), cleavage with cyanogen bromide, extraction of any acylhomoserine lactone into ethyl acetate, formation of a chemical derivative, and analysis by combined gas-liquid chromatography/mass spectrometry (GC/MS). The remaining cyanogen bromide fragments, insoluble in ethyl acetate, are available for further analysis by mass spectrometric or other methods if required. Using an acylhomoserine lactone labeled with a stable isotope as internal standard, the method is semiquantitative. It should be possible to develop a quantitative method if appropriate polypeptide standards are prepared. N-Terminal processing of eight recombinant-derived proteins is discussed.