Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes.

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol.

A continuous, spectroscopic analysis of the kinetics of elastase secretion by neutrophils. The dependence of secretion upon receptor occupancy.

We have developed a continuous spectroscopic method to analyze the kinetics of elastase secretion by human neutrophils. We have used the elastase-specific substrate methylsuccinylalanylalanylprolylvalylmethylcoumarin amide (Castillo, M. J.

A protease-like permeability factor in guinea pig skin: immunologic identity with plasma Hageman factor.

Vascular permeability enhancement activity of the protease-like permeability factor derived from guinea pig skin and of active guinea pig Hageman factor (beta HFa) were both inhibited by anti-guinea pig Hageman factor rabbit F(ab')2 antibody. The permeability activity of both factors was also absorbed on anti-Hageman factor F(ab')2-Sepharose beads. The latent form of the permeability factor derived from skin extracts produced a single immunoprecipitation line with anti-Hageman factor and gave a reaction of identity with a precipitation band developing between purified Hageman factor and anti-Hageman factor.

Studies on the pathogenesis of the adult respiratory distress syndrome.

Bronchoalveolar lavage (BAL) fluid was obtained from 24 sequentially studied patients with adult respiratory distress syndrome (ARDS) for assessment of potential activating and mediating factors. Proteolytic activity of the fluids was observed by measuring cleavage of radiolabeled proteins of the contact (Hageman factor) and complement systems. Proteolytic activity was observed in 17 of 24 (71%) patients with ARDS, and BAL fluid of the 7 ARDS patients without demonstrable, active, enzyme exhibited inhibitory activity for the proteolytic activity.

The role of parathyroidectomy in the management of hyperparathyroidism in patients on maintenance haemodialysis and after renal transplantation.

Between March 1964 and March 1980, 36 (34 dialysis, 2 transplant) of 327 patients accepted for the maintenance dialysis/transplantation programme at Charing Cross Hospital were submitted to parathyroidectomy. There were four main indications: persistent hypercalcaemia, progressive phalangeal erosions, aseptic necrosis of the femoral head and height loss with abnormal bone biopsy despite normal hand radiographs. At parathyroidectomy, 4 glands were removed in 1 patient, 3 1/2 glands in 24, 3 glands in 7, 2 glands in 3 and a single large gland in 1 patient.

Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes.

Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.

Ligand/receptor internalization: a spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human neutrophils.

We have compared the kinetics of the responses of neutrophils to the kinetics of ligand-receptor interaction and internalization, using as a model ligand the fluoresceinated hexapeptide N-CHO-Nle-Leu-Phe-Nle-Tyr-Lys-Fluorescein (Nle, norleucine). Cellular responses, ie, membrane depolarization, enzyme (elastase) secretion, and superoxide anion (O-2) generation, are all initiated within 10 sec of the exposure of cells to stimulus. In the cases of membrane depolarization and secretion (in cytochalasin B-treated cells), full responses are elicited by binding which occurs within 15 sec of peptide addition.

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies.

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation.

Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation.

Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide.

Guinea pig Hageman factor as a vascular permeability enhancement factor.

Hageman factor was purified from guinea pig plasma by successive column chromatography. The guinea pig Hageman factor appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 76,000 daltons by SDS--polyacrylamide gel electrophoresis and 105,000 daltons by gel filtration with a Sephadex G-150 column.

The distribution of lipopolysaccharide in normocomplementemic and C3-depleted rabbits and rhesus monkeys.

To examine the role of complement (C3) in determining the fate of lipopolysaccharide (LPS) in vivo, the distribution of LPS was studied in normocomplementemic (NC) and C3-depleted animals (pretreated with cobra venom factor [CoF]) after intravenous injection of highly purified, radioiodinated Salmonella minnesota R595 LPS. After injection of a lethal (250 micrograms) or nonlethal (5 micrograms) dose of LPS in NC and CoF rabbits and a lethal (5 mg/kg) dose of LPS in rhesus monkeys, the LPS disappeared from blood in a biphasic manner. In all cases, a substantial portion of the dose was removed from blood in an initial disappearance phase (t1/2 < 15 minutes), which, in some cases, was accelerated in CoF-treated animals.

Detection of active kallikrein in induced blister fluids of hereditary angioedema patients.

Six suction-induced blister fluids obtained from five patients with hereditary angioedema (HAE) contained active kallikrein, whereas only two blister fluids obtained from eight normal volunteers contained small amounts of this activity. Kallikrein was present in large amounts of HAE blister fluids as assessed by its ability to liberate smooth-muscle-contracting activity from purified high molecular weight kininogen. It was inhibited by purified antibodies specific for plasma prekallikrein and also by purified C1 inhibitor, but not by antibodies specific for C1s.

Dissemination of contact activation in plasma by plasma kallikrein.

The dissemination of contact activation of plasma was examined by measuring the cleavage of Hageman factor (HF) molecules on two separate sets of kaolin particles, one of which contained all of the components of the contact activation system, HF, prekallikrein (PK) and high molecular weight kininogen (HMWK) in whole normal plasma, and the second set of particles containing only HF and HMWK, being prepared with PK-deficient plasma. After mixing of the particles, cleavage of HF on the second set of particles occurred at a rate similar to that occurring on the first set of particles. This indicated that rapid dissemination and burst of activity of the contact reaction takes place in fluid phase.