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T Ganz

Publications and source records attributed to T Ganz.

At least 73 records · Page 4Linked to original sources
PubMed1997

Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems.

alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.

Blood Proteins↗
PubMed1997

Purification and characterization of defensins from cystic fibrosis sputum.

OBJECTIVE AND DESIGN: Cystic fibrosis (CF) sputum contains large numbers of neutrophils whose most abundant granule proteins are defensins. Within phagolysosomes, defensins kill microbes; however, extracellular defensins can be toxic to human cells. To begin to explore the possibility that defensins damage CF airways, this study examines the concentration and properties of defensins in CF sputum. MATERIALS: As a source of biological material in which to assay levels of defensins, purulent sputum was collected from persons with CF hospitalized with exacerbations of bronchitis. Purulent CF sputum was also a source of material for purification of defensins. METHODS: Defensin concentration in the cell-free component of CF sputum was measured by immunoassay. Defensins acid-extracted from sputum were purified by Sephacryl S-200 gel filtration chromatography and reverse phase-high pressure liquid chromatography (HPLC). Toxicity of CF defensins was tested by incubating the purified defensins with a line of CF tracheal cells cultured in serum five medium. RESULTS: In 5 patients with CF, sputum defensin levels ranged from 300 to > 1600 micrograms/ml, higher than levels previously reported in any human secretion or fluid and greatly exceeding concentrations toxic to mammalian cells in vitro. HPCL-purified CF sputum defensins were pure as judged by acid urea-PAGE and N-terminal sequencing, which revealed a mixture of defensins-1, -2 and -3 at ratios of approximately 4:2:1. At > 10 micrograms/ml the purified mixture was toxic for a line of CF tracheal cells cultured in serum-free medium, as judged by reductions in cell numbers and increased permeability to trypan blue. CONCLUSIONS: This study suggests that defensins in CF sputum are intact and sufficiently abundant that they may damage airway epithelium.

Blood Proteins↗
PubMed1997

Antimicrobial peptides of leukocytes.

Leukocytes use an array of antimicrobial peptides and proteins to help them destroy invading microorganisms. These endogenous antibiotic molecules are remarkable for their structural variety, rapid evolutionary divergence, intraspecies variation, and complex yet subtle interactions with their targets. This arsenal has been studied most extensively in neutrophils, where its members include lactoferrin, secretory phospholipase A2, lysozyme, and the cathelicidins in the secretory granule compartment; defensins, bactericidal permeability inducing protein, serprocidins, and (again) lysozyme in the azurophil granule compartment; and calprotectin in the cytosolic compartment. That antimicrobial peptides and proteins are not limited to neutrophils and other phagocytes was made clear by the recent discovery of a microbicidal protein, NK lysin, in porcine natural killer cells and cytotoxic T lymphocytes. The structural homology of NK-lysin to amebapore, an antimicrobial cytolytic peptide of the parasitic protozoan Entamoeba histolytica, provides remarkable support for a long-suspected evolutionary connection between the leukocytes of higher animals and their unicellular, protozoan ancestors.

Animals↗
PubMed1997

Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins.

Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors. Protegrins were stored in neutrophils as inactive proforms that were cleaved by neutrophil elastase to mature protegrins during the preparation of granule lysate and during phorbol myristate acetate-stimulated granule secretion from intact neutrophils. Recombinant pPG3 was efficiently cleaved by trace amounts of human neutrophil elastase or equivalent amounts of elastase activity from porcine neutrophils, but pPG3 was relatively resistant to porcine pancreatic elastase or human neutrophil cathepsin G. The recombinant pPG3 and neutrophil proprotegrins lacked microbicidal activity, but the mature protegrins generated in the elastase-mediated cleavage reaction were as active against Listeria monocytogenes as the chemically synthesized protegrin. The secretion and elastase-mediated activation of proprotegrins accounted for much of the stable microbicidal activity of porcine neutrophil secretions against L. monocytogenes. Secreted proprotegrins and trace amounts of elastase constitute a binary microbicidal system that is likely to contribute to the antimicrobial activity of porcine inflammatory fluids.

Amino Acid Sequence↗
PubMed1997

Localization of human intestinal defensin 5 in Paneth cell granules.

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.

Adult↗
PubMed1997

Broad-spectrum antimicrobial activity of human intestinal defensin 5.

Defensins are antibiotic peptides expressed in human and animal myeloid and epithelial cells. Due to the limited availability of natural peptides, the properties of human epithelial defensins have not been studied. We assayed the microbicidal activity of recombinant human intestinal defensin 5 (rHD-5) in the presence of salt (O to 150 mM NaCl) with varied pH (pH 5.5 to pH 8.5) and trypsin (25 and 250 microg/ml). rHD-5 exhibits microbicidal activity against Listeria monocytogenes, Escherichia coli, and Candida albicans. In contrast to cryptdins, the mouse intestinal defensins, rHD-5 is active against both mouse-virulent wild-type Salmonella typhimurium and its isogenic, mouse-avirulent phoP mutant. In the presence of salt, rHD-5 activity was reduced, and at 100 mM NaCl, activity against S. typhimurium was abolished. However, at all salt concentrations tested, rHD-5 remained bactericidal to L. monocytogenes. Activity against L. monocytogenes was not pH dependent but was diminished at pH 5.5 against wild-type S. typhimurium. This acid-induced resistance may have been mediated by the virulence gene regulator phoP, since the phoP mutant was equally sensitive at pH 5.5 and pH 7.4. In the presence of trypsin, rHD-5 was partially cleaved, but even then, rHD-5 at 100 microg/ml decreased the number of CFU of wild-type S. typhimurium by more than 99%. The persistence of microbicidal activity of rHD-5 under these conditions supports the notion that naturally occurring human intestinal defensin is an effective arm of mucosal host defense.

Animals↗
PubMed1997

Immunohistochemical localization of defensin in human coronary vessels.

Neutrophil defensins comprise a family of cationic peptides that possess potent antimicrobial activity. Defensins are normally sequestered in cytoplasmic granules with their primary site of action in phagolysosomes, although some peptide is released into the circulation during the course of infection or inflammation. In view of the fact that neutrophils adhere to the endothelium and that defensins have been reported to bind to human endothelial cells in vitro, we used immunohistochemistry to study the distribution of these peptides in normal and in atherosclerotic human coronary arteries. Defensin was found primarily in the intima of normal and atherosclerotic vessels, most prominently in association with intimal smooth muscle cells. Both large- and small-vessel endothelium stained focally for defensin. Defensin was also found in the media near the external elastic lamina and in some periadventitial vessels. The same distribution was seen in vessels that had been perfusion fixed immediately upon procurement, excluding diffusion of defensin from PMNs ex vivo. These data indicate that neutrophil defensin is present in the walls of human coronary arteries. The deposition of defensin in vessels may contribute to the pathophysiological consequences of inflammation in addition to their role in host defense.

Anti-Infective Agents↗
PubMed1997

Antimicrobial peptides of phagocytes and epithelia.

Human and other vertebrate leukocytes contain multiple distinct antimicrobial (poly)peptides. Of these, BPI is a LPB protein active against gram-negative bacteria, PLA2 specifically cleaves bacterial phospholipids, while defensins and cathelicidins are broad spectrum antimicrobials that preferentially permeabilize microbial membranes. These and other polypeptides function in both phagocytic and extracellular killing of microbes, attacking multiple molecular targets to cooperatively penetrate and disrupt the microbial surfaces and membrane barriers. Such antimicrobial substances are of interest not only for students of phagocytic and epithelial host defenses but may lead to the development of novel pharmaceuticals for the treatment of infections and their sequellae.

Animals↗
PubMed1996

Endogenous vertebrate antibiotics. Defensins, protegrins, and other cysteine-rich antimicrobial peptides.

Although newly recognized, endogenous cystine-stabilized beta-sheet antimicrobial peptides have ancient origins. These peptides can arm circulating phagocytes and cells of the gastrointestinal, respiratory, and genitourinary tracts to resist invasion by bacteria, mycobacteria, fungi, and enveloped viruses. Defensins and protegrin-like peptides are likely to play a considerable role in innate immunity and may provide molecular templates that can be used to generate novel antibiotics for topical and systemic use.

Amino Acid Sequence↗
PubMed1996

Defensin modulates tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells.

Defensins are naturally occurring antimicrobial peptides that may participate in host defense against microorganisms. We previously reported that the amino acid sequence of leukocyte defensins resembles the lysine-binding site in the kringles of plasminogen and that defensin inhibits fibrinolysis mediated by tissue-type plasminogen activator (tPA) and plasminogen. In the present paper we analyze the mechanisms of this inhibition. Defensin binds specifically to cultured human umbilical vein endothelial cells (HUVEC) (half-maximal binding = 3 microM) as well as to fibrin. At saturating concentrations (5-10 microM), defensin stimulates the maximum binding of plasminogen to HUVEC and to fibrin approximately 10-fold. However, defensin inhibits plasminogen binding to both surfaces at concentrations >10 microM. Defensin also inhibits tPA and plasminogen-mediated fibrinolysis in a dose-dependent manner at all concentrations tested. Fibrinolysis is almost totally inhibited by 6 microM defensin, a concentration that stimulates the binding of plasminogen to fibrin. Discordance between the enhancement of plasminogen binding and its activation cannot be explained by an inhibitory effect of defensin on tPA binding nor by inhibition of plasmin activity, each of which occur only at higher concentrations. Rather, these results suggest that plasminogen bound to fibrin in the presence of defensin is less susceptible to activation by tPA.

Anti-Infective Agents↗
PubMed1996

Intramolecular inhibition of human defensin HNP-1 by its propiece.

We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.

Amino Acid Sequence↗
PubMed1995

Structures of genes for two cathelin-associated antimicrobial peptides: prophenin-2 and PR-39.

We characterized genes for prophenin (PF)-2 and PR-39, two cathelin-associated antimicrobial peptides found in porcine leukocytes. Both contained 4 exons and 3 introns and were compact, contiguous and highly homologous. Exons I-III encoded most of their cathelin domains. Exon IV specified the final few cathelin residues, including its conserved C-terminal valine, followed by the mature PR-39 peptide or a PF-2 precursor. The highly conserved 5' flanking sequences of this gene family contained NF-kappa B, IL-6, GM-CSF and NF-1 binding motifs and the introns were unusually conserved. These data suggest that the panoply of porcine cathelin-associated antimicrobial peptides arose relatively recently via gene reduplications and exon shuffling, and that in vivo expression of cathelin-associated antimicrobial peptides may respond to mediators generated early during infection.

Amino Acid Sequence↗
PubMed1995

The structure of porcine protegrin genes.

We cloned the genes of three protegrins, a family of cathelin-associated antimicrobial peptides originally isolated from porcine leukocytes. Each gene comprised 4 exons and 3 introns, wherein Exon I encoded the signal sequence and the first 37 amino acids of cathelin, Exons II and III contained 36 and 24 additional cathelin residues and Exon IV contained the final two cathelin residues followed by the protegrin sequence. This quadripartite gene structure helps explain how structurally diverse antimicrobial peptides can be expressed on common, cathelin-containing precursors. Southern blot probed with an oligonucleotide specific for protegrin genes suggested that several identical or nearly identical protegrin genes were densely clustered in the pig chromosome.

Amino Acid Sequence↗
PubMed1995

Murine 32D c13 cells--a transfectable model of phagocyte granule formation.

Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasmic granules and delivered by fusion to the phagocytic vacuoles (phagosomes). To determine if the murine myeloid cell line 32D c13 is suitable as a genetic model of protein translocation to granules and phagosomes, we permanently transduced 32D c13 cells with human HNP-1 defensin cDNA, incubated them with opsonized zymosan, and immunostained them for human defensin HNP-1. Although their phagocytic rate was much slower than that of neutrophil granulocytes, 32D c13 cells ingested zymosan into vacuoles that accumulated most of the transgenic defensin. The 32D c13 cell line should be useful for studies of the targeting of proteins to granules and phagosomes.

Animals↗
PubMed1995

The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting.

Human defensins (human neutrophil peptides) HNP 1-3 are 29-30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44-45 aa anionic propiece. To study the role of the propiece, we constructed five in-frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21-64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24-hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21-28 and delta 21-38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21-51 and delta 21-64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40-51, which contains highly conserved secondary structure. The propiece segment 40-51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.

3T3 Cells↗
PubMed1995

Defensins and other endogenous peptide antibiotics of vertebrates.

Gene-encoded peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Their de novo synthesis or release from storage sites can be induced rapidly, which makes them particularly important in the initial phases of resistance to microbial invasion. The endogenous antimicrobial peptides of animals are products of single genes and are synthesized as preproproteins. Multistep processing yields the mature peptide, which generally acts by inducing microbial membrane permeabilization. Several families of antimicrobial peptides have been identified that differ with respect to the presence of disulfide linkages, amino acid composition, structural conformation, and spectrum of activity. The arginine-rich three disulfide-containing beta-sheet defensins are remarkably abundant and widely distributed in animals and plants. The antibiotic peptides of higher eukaryotes merit further study for their role in natural immunity and their potential as novel therapeutic compounds.

Amino Acid Sequence↗
PubMed1995

Defensins.

Defensins are a family of small cationic, antibiotic peptides that contain six cysteines in disulfide linkage. The peptides are abundant in phagocytes and small intestinal mucosa of humans and other mammals and in the hemolymph of insects. They contribute to host defense against microbes and may participate in tissue inflammation and endocrine regulation during infection. Bioengineered defensins are potentially useful as prophylactic and therapeutic agents in infections.

Amino Acid Sequence↗