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Biomedical subjects

B U Wintroub

Publications and source records attributed to B U Wintroub.

At least 55 records · Page 3Linked to original sources
PubMed1982

The granulocyte-angiotensin system. Angiotensin I-converting activity of cathepsin G.

Cathepsin G, an Mr = 26,000-29,000 cationic human neutrophil lysosomal serine protease, releases angiotensin II from angiotensinogen and was, therefore, examined for angiotensin I-converting activity. Cathepsin G-dependent angiotensin I conversion was detected by a high performance liquid chromatography assay which permitted independent quantitation of angiotensin I and angiotensin II and detection of angiotensin degradation products. 1.8-5.0 X 10(-9) M cathepsin G converted angiotensin I (3.3 X 10(-4) M) to angiotensin II without further degradation of angiotensin II. The pH optimum for cathepsin G-catalyzed angiotensin I conversion was pH 7.0-7.5, and the Km and Kcat were 2.2 X 10(-4) M and 3.4 s-1, respectively. In contrast to dipeptidyl hydrolase-converting enzyme, cathepsin G did not inactivate bradykinin, did not cleave hippuryl-His-Leu, and was not inhibited by 10(-4) M Captopril or SQ 20881. Purified human neutrophils stimulated with 2.5 X 10(-6) M-10(-10) M fMet-Leu-Phe released angiotensin-converting activity with a Km of 3.3 X 10(-4) M. That the angiotensin-converting activity released from neutrophils was attributable to cathepsin G was indicated by similar susceptibility to inhibitors and adsorption by goat antibody to cathepsin G. The granulocyte-angiotensin system provides a mechanism for the local generation of angiotensin II at sites of neutrophil accumulation and may be of significance in regulation of blood flow in tissue microvasculature.

Angiotensin I↗
PubMed1982

Bullous pemphigoid, an ultrastructural study of the inflammatory response: eosinophil, basophil and mast cell granule changes in multiple biopsies from one patient.

We have studied by electron and light microscopy the inflammatory reaction in lesions at various stages of clinical development from a patient with bullous pemphigoid. The evolution of clinical lesions was associated with a sequence of histopathologic events which began with alterations of mast cells and proceeded to infiltration, first with lymphocytes and later with eosinophils and basophils. Mast cells in the papillary and reticular dermis demonstrated a unique, focal, irregular loss of granule contents. Intact eosinophils demonstrated intracytoplasmic losses of granule contents and karyorrhectic and karyolytic eosinophils had released membranebound granules. Partially and completely degranulated basophils were present within a fibrin gel which formed in the dermis. Thus, the sequence of histopathologic events in the pathogenesis of bullous pemphigoid includes mast cell granule alterations and release of granule contents from eosinophils which are undergoing nuclear and cytoplasmic damage.

Basophils↗
PubMed1982

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

A human neutrophil protease, initially termed neutral peptide-generating protease, has been shown to cleave angiotensin II directly from angiotensinogen and has been identified as leukocyte cathepsin G. When purified neutrophils were disrupted by nitrogen cavitation and fractionated by differential centrifugation, 44 and 24% of the angiotensin II-generating activity was in the lysosomal and undisrupted cell fractions, respectively. Cytochalasin B-treated human neutrophils stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine released beta-glucuronidase, lysozyme, and angiotensin II-generating protease in a dose-dependent fashion, consistent with localization of this protease to the neutrophil granule. Individually purified angiotensin II-generating protease and cathepsin G had similar proteolytic and esterolytic activity for angiotensinogen and N-benzoyl-L-tyrosine ethyl ester on a weight basis, exhibited identical mobilities by SDS-gradient polyacrylamide gel electrophoresis and pH 4.3 disc-gel electrophoresis, and gave precipitin lines of antigenic identity on Ouchterlony analysis with goat antibody to the angiotensin II-generating protease. Thus, the angiotensin II-generating protease of human neutrophils has been identified as cathepsin G on the basis of subcellular localization, substrate specificity, physicochemical characteristics, and antigenic identity.

Angiotensin II↗
PubMed1981

A human neutrophil-dependent pathway for generation of angiotensin II: purification and physicochemical characterization of the plasma protein substrate.

Human neutrophils contain a neutral protease, previously designated "neutral peptide-generating protease," which generates a smooth muscle contractile activity from a plasma protein substrate. The plasma protein substrate has been purified to homogeneity from fresh citrated human plasma by 45% (wt/vol) ammonium sulfate precipitation of contaminating proteins, Affi-Gel Blue affinity chromatography, hydroxylapatite chromatography, phenyl-Sepharose hydrophobic chromatography, and Sephacryl S-200 gel filtration. The purified product produced a single stained protein on alkaline disc gel electrophoresis and elicited a monospecific goat antiserum. Purification was approximately 330- to 350-fold, and overall recovery was 6-11% of substrate protein in starting plasma as determined by quantitative radial immunodiffusion assay. The substrate has an isoelectric point of pH 4.6-5.1 and is a single polypeptide chain glycoprotein of Mr 62,000-67,000 by sodium dodecyl sulfate electrophoresis. The mean(+/- SD) concentration of this plasma protein substrate in normal serum is 120 +/- 22 microgram/ml. The plasma protein substrate of the neutrophil neutral protease may be identical to human angiotensinogen (renin substrate) because the physicochemical characteristics are similar and the peptide product is recognized by antibody to angiotensin II.

Angiotensin II↗
PubMed1981

A human neutrophil-dependent pathway for generation of angiotensin II. Purification of the product and identification as angiotensin II.

A human neutrophil lysosomal protease interacts at physiologic pH with a 62,000--67,000-mol wt plasma protein substrate to generate a vasoactive, smooth muscle-contracting "neutral" peptide. The peptide product of this system, previously designated the "neutral" peptide-generating pathway, was generated from purified components and purified by Bio-Gel P2 gel filtration and reverse-phase high performance liquid chromatography with a 50--60% yield of starting activity. The purified peptide had an amino acid composition of Asx, Pro, Val, Ile, Tyr, Phe, His, Arg, a composition identical to that of angiotensin II. The peptide and synthetic angiotensin II each filtered at 48--52% bed volume on Bio-Gel P2, had an isoelectric point of Ph 7.8--8.1 at 4 degrees C, migrated 3 cm toward the cathode during pH 6.4 low-voltage paper electrophoresis, and had a retention time of 44.8 min during reverse-phase high-performance liquid chromatography. In addition, the functional activity of the peptide at each purification step correlated with angiotensin II content determined by specific radioimmunoassay. The amino acid sequence of 25 nmol of the peptide was Asp-Arg-Val-Try-Ile-His-Pro-Phe, the same covalent structure as that of angiotensin II. Therefore, under physiologic conditions, in the absence of renin or angiotensin converting enzyme, a human neutrophil neutral protease cleaves a plasma protein to yield angiotensin II. This pathway provides a mechanism through which the neutrophil may alter local blood flow during inflammation by generation of a potent vasoactive peptide.

Amino Acids↗
PubMed1980

Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease.

The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Purification and characterization of a human neutrophil neutral protease. The neutral peptide-generating protease.

A human neutrophil neutral protease which generates a low molecular weight peptide from a plasma protein substrate and cleaves the basic amino acid ester substrates alpha-N-p-tosyl-l-arginine methyl ester HCl, alpha-N-benzoyl-l-arginine-methyl ester HCl, and alpha-N-carbobenzoxy-l-lysine-p-nitrophenyl ester has been purified to homogeneity and distinguished from the known lysosomal neutrophil proteases. The starting activity was obtained from purified human neutrophils by homogenization, sedimentation by low-speed centrifugation, and high salt elution of the insoluble material. Purification was achieved by aprotinin-affinity chromatography, precipitation at low ionic strength, and gel filtration. The overall recovery, relative to the activity in the starting eluate of the neutrophil fraction, was congruent with50% with a 200- to 400-fold increase in specific activity. After treatment with diisopropylfluorophosphate to eliminate autodegradation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced and unreduced protein gave a single protein band of 29,000-30,000 mol wt. The isoelectric point determined in sucrose gradients ranged from pH 7.8 to 8.3 with a peak at pH 8.0. This neutrophil protease, like cathepsin G and elastase, is composed of a single polypeptide chain of congruent with30,000 mol wt, but differs from cathepsin G and elastase in its less cationic isoelectric point and its failure to cleave synthetic substrates presenting an aromatic amino acid ester linkage and alanyl peptide bonds, respectively.

Chromatography, Affinity↗
PubMed1978

Morphologic and functional evidence for release of mast-cell products in bullous pemphigoid.

We studied nine patients with bullous pemphigoid, a generalized cutaneous eruption- for evidence of mast-cell involvement during development of lesions. As in other reports, six of nine patients demonstrated a serum antibody directed against the epidermal basement-membrane zone. Direct immunofluorescence studies of lesions revealed depostion of immunoglobulin and complement proteins at the basement-membrane zone in six of nine and nine of nine patients, respectively. Participation of mast cells was suggested by a sequence of pathologic alterations in which there was progressive mast-cell degranulation and late eosinophil infiltration. In addition, a factor chemotactic for human eosinophils with the size and charge characteristics of the eosinophil chemotactic factor of anaphylaxis was identified in blullous fluid. The data indicate that, in addition to activation of the complement system, involvement of mast cells is an early and continuing event in the development of the cutanenous lesions of bullous pemphigoid.

Autoantibodies↗
PubMed1977

A neutrophil-dependent pathway for the generation of a neutral peptide mediator. II. Subcellular localization of the neutrophil protease.

The human neutrophil neutral peptide-generating protease was associated with the plasma membrane marker 5'-nucleotidase on sucrose density gradient centrifugation of sonicates of granule-free fractions following homogenization and velocity sedimentation. The two activities were also associated on sucrose density gradient fractionation of plasma membranes obtained by hypotonic lysis in EDTA containing buffers, a technique which minimizes aggregation. Treatment of fractions containing these enzymatic activities with 1-0 M NaCl separated the neutral peptide-generating proteasein to the eluate while leaving the 5'-nucleotidase in the pellet. Gel filtration of the solubilized neutral peptide-generating protease through Sephadex G-100 in 1-0 M NaCl demonstrated that the protease had an approximate mol. wt of 20,000 while filtration in physiological salt concentrations yielded activity only in the excluded volume. In both cases, there was complete recovery of neutral peptide-generating activity suggesting that the filtration characteristics of the protease were determined by the salt concentration. The solubilized purified protease, the whole cell sonicates, and the intact cells interacted with heat-inactivated plasma to yield the same product, a neutral peptide with a 1000 molecular weight and an isoelectric point of 7-2-7-6. The neutral peptide-generating protease in each instance was inhibited in dose-response fashion by alpha-1-antitrypsin, LBTI, and DFP. Only 30-60% of the protease sites were functional on intact cells as revealed by substrate cleavage or were available to inhibitors. The neutrophil protease which generates neutral peptide is an extrinsic plasma membrane protein with an approximate mol. wt of 20,000 which functions as an ectoenzyme.

Cell Fractionation↗
PubMed1976

A phagocytosis-enhancing factor in human plasma.

A phagocytosis-enhancing factor (PEF) with the capacity to stimulate the ingestion of sensitized sheep erythrocytes by human polymorphonuclear and mononuclear leucocytes has been isolated from human plasma by chromatography on DEAE-cellulose and filtration on Sephadex G-150 and Sephadex G-100. PEF is a protein of approximately 70,000 molecular weight which is susceptible to inactivation by heating at 60 degrees or by tryptic digestion. PEF promotes phagocytosis of erythrocytes sensitized with intact 7S antibody or bearing the C3b complement fragment, but not of unsensitized erythrocytes or erythrocytes sensitized with 19S antibody. The specificity of PEF interaction with target erythrocytes and the persistence of its stimulatory effect after the target cells are washed suggest that it promotes phagocytosis by an action on the erythrocytes.

Antibodies↗
PubMed1974

A neutrophil-dependent pathway for the generation of a neutral peptide mediator: partial characterization of components and control by alpha-1-antitrypsin.

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an alpha-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and alpha-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to alpha-1-antitrypsin was established both by inhibition with highly purified alpha-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) alpha-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) alpha-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide.

Animals↗
PubMed1969

The pulmonary and systemic circulatory response to dopamine infusion.

1. The pulmonary and systemic circulatory responses to dopamine infused at 8, 15, 25 and 30 mug/kg per min were studied in eight mongrel dogs.2. Mean pulmonary artery pressure, mean left atrial pressure, mean aortic pressure, mean aortic flow and the electrocardiogram were monitored in openchest preparations. Pulmonary vascular resistance, systemic vascular resistance and stroke volume were calculated.3. Significant increases in mean pulmonary and aortic pressures were noted at dopamine infusions of 25 and 30 mug/kg per min. The left atrial pressure fell significantly at 15 mug/kg per min and rose significantly at 30 mug/kg per min. Mean aortic flow increased at all four doses, while heart rate showed no change. Pulmonary vascular resistance did not change significantly at any dose level, but systemic vascular resistance fell slightly at 8 and 15 mug/kg per min and rose significantly at 30 mug/kg per min. Stroke volume was significantly elevated at infusions of 25 and 30 mug/kg per min.4. The systemic circulatory response to dopamine is similar to that described by previous investigators.5. The increased pulmonary pressures without change in resistance suggest a dopamine-induced increase in smooth muscle tension in the pulmonary vasculature.

Animals↗