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T Ganz

Publications and source records attributed to T Ganz.

At least 109 records · Page 6Linked to original sources
PubMed1992

Posttranslational processing of defensins in immature human myeloid cells.

Human neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL-60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defensins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloroquine, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity.

Amino Acid Sequence↗
PubMed1992

Defensins: endogenous antibiotic peptides from human leukocytes.

A variety of endogenous antimicrobial peptides equip mammals, amphibians, insects and plants to defend themselves against microbial pathogens. Defensins are small peptides of mammalian cells that contain 29-35 amino acid residues, including six invariant cysteines that form three intramolecular disulphide bonds. They are produced by the sequential proteolysis of precursors that contain approximately 95 amino acids and are synthesized by several types of cells, especially the bone marrow precursors of blood neutrophils. Defensins constitute 5-7% of the total protein in human neutrophils and are present in high concentration in the azurophil granules and phagocytic vacuoles. The production of defensins by human neutrophils amounts to approximately 10 mg/kg body weight per day. In certain mammalian species lung macrophages and specialized epithelial (Paneth) cells in the small intestine also produce defensins. Defensins are complexly folded, amphipathic, rich in antiparallel beta-sheet but devoid of alpha-helical domains. Their unusually broad antimicrobial spectrum encompasses Gram-positive and Gram-negative bacteria, many fungi, mycobacteria, spirochetes and several enveloped viruses. The antimicrobial properties of defensins result from their insertion into target cell membranes and the formation of voltage-sensitive channels. Given their abundance and broad spectrum of antimicrobial activity, defensins may contribute substantially to innate resistance to infection.

Amino Acid Sequence↗
PubMed1992

Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity?

Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP-3a, NP-4, and NP-5. The preprodefensins NP-3a, NP-4, and NP-5, like the previously characterized preprodefensins, lack consensus sequences for N-linked glycosylation, suggesting that defensins are targeted to lysosome-like granules by a mechanism not dependent on the mannose-6-phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.

Amino Acid Sequence↗
PubMed1992

Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare.

We have examined the activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare. M. avium-M. intracellulare at 2.5 x 10(6)/ml or 2.5 x 10(8)/ml was cultured in the presence of defensins at 37 degrees C from 4 to 48 h. After incubation, CFU were enumerated. Human neutrophil peptide 1 (HNP-1) at 5 micrograms/ml had the ability to kill M. avium-M. intracellulare. Treatment with HNP-1 resulted in significant (96.3 to 97.7%) killing of M. avium-M. intracellulare, even after taking clumping into consideration. This activity was not affected by the presence of calcium (0.5 and 1.0 mM), magnesium (0.5 and 1.0 mM), or sodium chloride (25, 50, and 100 mM). The optimal pH for bactericidal activity was higher than 5. We tested numerous M. avium-M. intracellulare strains, and HNP-1 was successful in killing every strain, although the degree of killing varied among them (34.2 to 87.2%). Additionally, this activity was independent of colonial morphology. We also examined the activity of HNP-2 and HNP-3 against M. avium-M. intracellulare and found that they were as effective in killing M. avium-M. intracellulare as HNP-1 was. These observations suggest that defensins may play an important role in the host defense against M. avium-M. intracellulare.

Blood Bactericidal Activity↗
PubMed1991

An enzyme immunoassay for human defensins.

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces. The assay has a sensitivity of 0.04-0.05 ng/ml and a working range of 0.05-10 ng/ml.

Antibodies, Monoclonal↗
PubMed1991

Contribution of rabbit leukocyte defensins to the host response in experimental syphilis.

In the companion paper (L. A. Borenstein, M. E. Selsted, R. I. Lehrer, and J. N. Miller, Infect. Immun. 59:1359-1367, 1991), we report that rabbit alveolar macrophage and neutrophil derived defensins possess antimicrobial activity against Treponema pallidum subsp. pallidum, the etiologic agent of syphilis. In this study, antisera specific for NP-1 and NP-2 (defensins present in certain macrophages and polymorphonuclear leukocytes) and NP-5 (a defensin produced only in neutrophils) were used to detect these peptides by immunoperoxidase staining in testicular lesions from infected rabbits. Profound amounts of cell-free and cell-associated defensins were detected in the tunica albuginea and interstitial spaces during the first 24 h of infection. The presence of defensins was transient and almost undetectable by day 4. Interstitial defensins were detected again at day 10 and increased through day 16, at which time lesion healing was evident by hematoxylin and eosin staining. The appearance and increase in detectable defensins between days 10 and 16 of infection correlated with a reduction in numbers and disappearance of T. pallidum, as demonstrated by using silver staining. The extent and pattern of immunostaining for NP-1 and NP-2 corresponded with immunostaining for NP-5 and identified neutrophils as the cellular source of the defensins. These findings indicate that defensins may contribute to the control of local T. pallidum infection and suggest a role for acute inflammatory processes in the resolution of early experimental syphilis.

Animals↗
PubMed1991

Activated alpha 2-macroglobulin is a principal defensin-binding protein.

Defensins are highly abundant and variably cationic peptides that possess antimicrobial, cytotoxic, and chemoattractant properties and equip mammalian phagocytes for participation in host defense and inflammatory processes. We studied the binding of the human defensin HNP-1 by proteins in plasma and serum and identified activated (F-form) alpha 2-macroglobulin (alpha 2M) as a principal binding protein for HNP-1. In contrast, native (S-form) alpha 2M bound little HNP-1. The binding of HNP-1 by F-form alpha 2M was resistant to salt and boiling in 2% sodium dodecyl sulfate but was ablated by dithiothreitol. Pretreatment of methylamine-activated serum or plasma by iodoacetamide substantially decreased the binding of HNP-1 to alpha 2M, suggesting that thiol groups in activated alpha 2M play a role in defensin binding, possibly by covalently trapping defensins via thiol-disulfide exchange. Western blots of conventionally collected sera showed endogenous defensins complexed with the F-form of alpha 2M, indicating that the generation of defensin-alpha 2M complexes was not limited to the in vitro model of methylamine-activated serum or plasma and radiolabeled HNP-1. Previous studies indicated that native alpha 2M can be converted to its F-form by many proteases, including those released by neutrophils and platelets, and that the F-form is recognized and internalized by specific receptors on macrophages and hepatocytes. Our findings suggest that the alpha 2M system may function as a scavenger of defensins and other peptide mediators in inflamed tissues and may constitute an important mechanism for the regulation and containment of inflammation.

Blood Bactericidal Activity↗
PubMed1990

Purification and characterization of a major phosphatidylserine-binding phosphoprotein from human platelets.

We describe the isolation, lipid-binding properties and partial amino acid sequence of PS-p68, a novel 68 kDa phosphatidylserine-binding protein from human platelets. PS-p68 is an abundant constituent of platelets, accounting for 0.5-0.75% of total cell protein. It was purified from platelet cytosol by affinity chromatography. Amino acid sequence analysis yielded no similarity to identified proteins. In contrast with most known phospholipid-binding proteins, PS-p68 does not bind Ca2+ and does not require Ca2+ for its binding of phosphatidylserine. Phosphatidylserine binding to PS-p68 was inhibited by phosphatidic acid and by alkylphospholipids. PS-p68 was isolated as a major phosphoprotein from 32P-labelled platelets and was found to function as a protein kinase C substrate in vitro. However, treatment of intact platelets with phorbol 12-myristate 13-acetate, thrombin or carbacyclin did not increase PS-p68 phosphorylation. Platelets appear to be the only blood cells containing PS-p68, which was not detected in neutrophils, monocytes and lymphocytes.

Amino Acid Sequence↗
PubMed1990

A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease.

Antineutrophil cytoplasmic antibodies (ANCAs) have recently been demonstrated to be of importance in Wegener's granulomatosis and certain other forms of vasculitis associated with glomerulonephritis. With a fixed-cell ELISA, we demonstrated that ANCAs occur in the serum of patients with inflammatory bowel disease (IBD) involving the colon. In a blinded study, sera from 21 of 25 patients with ulcerative colitis (UC) and five of 25 patients with Crohn's disease had binding in the fixed-cell ELISA. The five reactive sera from patients with Crohn's disease were associated with the presence of clear colonic involvement. The presence of ANCA in patients with UC was not influenced by disease distribution or activity. Indeed, such antibodies were present in four subjects with UC more than 5 years after colectomy. The IBD-associated ANCAs were distinct from ANCAs reported in patients with Wegener's granulomatosis since the pattern of staining on indirect immunofluorescence exhibited a nongranular perinuclear distribution (P-ANCA). The P-ANCA observed in IBD did not react with myeloperoxidase and thus was distinct from the P-ANCA observed in vasculitis with cresentric glomerulonephritis. IBD and, in particular, UC, is associated with a distinct subset of P-ANCA, which may have important diagnostic and potential pathophysiologic implications.

Adult↗
PubMed1990

Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes.

Defensins are cationic, cysteine-rich peptides (Mr = 3500-4000) found in the cytoplasmic granules of neutrophils and macrophages. These peptides possess broad antimicrobial activity in vitro against bacteria, fungi, tumor cells, and enveloped viruses, and they are believed to contribute to the "oxygen-independent" antimicrobial defenses of neutrophils and macrophages. Pathophysiologic studies in vitro have pointed to the plasma membrane as a possible target for the cytotoxic action of defensins. We report here that defensins form voltage-dependent, weakly anion-selective channels in planar lipid bilayer membranes, and we suggest that this channel-forming ability contributes to their antimicrobial properties observed in vitro.

Anti-Bacterial Agents↗
PubMed1990

Killing of oral, gram-negative, facultative bacteria by the rabbit defensin, NP-1.

Oral, gram-negative, facultative bacteria, including Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. have been associated with destructive periodontal infection. Neutrophils play a critical role in defending the periodontium against destructive infection. Defensins are antimicrobial peptides that have been isolated in human, rabbit, guinea pig, and rat leukocytes that may constitute an important nonoxidative mechanism of killing. The purpose of this study was to examine the sensitivity of a battery of oral, gram-negative, facultative bacteria to the bactericidal effects of the isolated rabbit peptide NP-1. All species tested were killed by NP-1; however, there was strain-to-strain variation in sensitivity. The bactericidal effect was not dependent on net bacterial growth, although metabolic activity was evident as assessed by bacterial oxygen consumption. We conclude that bacteria are sensitive to the cidal mechanism involved in defensin-mediated bacterial killing and that the conditions of this assay system support the killing of bacteria by the defensin peptides.

Actinobacillus↗
PubMed1990

Defensins.

Defensins are a family of small, variably cationic proteins which are highly abundant in the granules of mammalian phagocytes. Three defensins, HNP-1, 2, and 3, comprise 30-50% of total protein in azurophil granules of human neutrophils. Some defensins are broadly antimicrobial, antiviral and cytotoxic, while others are chemotactic, opsonic, or may modulate hormonal responses. The defensin molecule typically consists of 29-34 amino acids with a conserved pattern of disulfide linkage among its 6 cysteines. The three-dimensional fold of defensins forms a highly amphiphilic molecule. Microbicidal and cytotoxic properties of defensins are most likely a consequence of their ability to insert into biological membranes and to generate pores. Defensins are synthesized by phagocytes or their precursors as a 94-95 amino acid charge-neutralized preprodefensin, an arrangement which may avoid cytotoxic injury to the phagocyte. Although defensins were recognized only recently, the existence of homologs in certain invertebrates suggests that they are ancestral components of the host defense system.

Amino Acid Sequence↗
PubMed1990

Polymorphic expression of defensins in neutrophils from outbred rats.

We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7. Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans. The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4. This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others. These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents.

Amino Acids↗
PubMed1990

In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

Blood Bactericidal Activity↗