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B Agerberth

Publications and source records attributed to B Agerberth.

At least 37 records · Page 2Linked to original sources
PubMed1997

The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.

The epithelia constitute a major barrier to the environment and provide the first line of defense against invading microbes. Antimicrobial peptides are emerging as participants in the defense system of epithelial barriers in general. Originally we isolated the human antimicrobial peptide LL-37 from granulocytes. The gene (CAMP or cathelicidin antimicrobial peptide) coding for this peptide belongs to the cathelicidin family, whose members contain a conserved pro-part of the cathelin type. The human genome seems to have only one gene of this family, whereas some mammalian species have several cathelicidin genes. In the present work we demonstrate up-regulation of this human cathelicidin gene in inflammatory skin disorders, whereas in normal skin no induction was found. By in situ hybridization and immunohistochemistry the transcript and the peptide were located in keratinocytes throughout the epidermis of the inflammatory regions. In addition, the peptide was detected in partially pure fractions derived from psoriatic scales by immunoblotting. These fractions also exhibited antibacterial activity. We propose a protective role for LL-37, when the integrity of the skin barrier is damaged, participating in the first line of defense, and preventing local infection and systemic invasion of microbes.

Adult↗
PubMed1996

The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.

The peptide FA-LL-37, previously termed FALL-39, was originally predicted from on ORF of a cDNA clone isolated from a human bone marrow library. This peptide was synthesized and found to have antibacterial activity. We have now characterized and sequenced the complete gene for FA-LL-37, termed FALL39. It is a compact gene of 1963 bp with four exons. Exons 1-3 code for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Our results indicate that FALL39 is the only member of the cathelin gene family present in the human genome. Potential binding sites for acute-phase-response factors are identified in the promoter and in intron 2. A possible role for the cytokine interleukin-6 in the regulation of FALL 39 is discussed. Anti-(FA-LL-37) IgG located the peptide in granulocytes and we isolated the mature peptide from these cells after degranulation. Structural analysis determined the mature peptide to be LL-37. To obtain LL-37 for antibacterial assays, synthetic FA-LL-37 was degraded with dipeptidyl-peptidase I. This analysis showed that mature LL-37 is a potent antibacterial peptide.

Amino Acid Sequence↗
PubMed1996

Biochemical and antibacterial analysis of human wound and blister fluid.

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11,1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

Adult↗
PubMed1996

PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart.

The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13-34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.

Animals↗
PubMed1996

NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells.

NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.

Animals↗
PubMed1995

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.

Amino Acid Sequence↗
PubMed1995

FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.

PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.

Adult↗
PubMed1994

Secondary structure and membrane interaction of PR-39, a Pro+Arg-rich antibacterial peptide.

PR-39 is a 4719-Da peptide isolated from pig intestine and belonging to the recently discovered family of Pro+Arg-rich antibacterial peptides. PR-39 does not lyse Escherichia coli, instead the lethal action is probably linked to the termination of DNA and protein synthesis [Boman, H. G., Agerberth, B. & Boman, A. (1993) Infect. Immun. 61, 2978-2984]. Circular dichroism and Fourier-transform infrared spectroscopy have been used to investigate the secondary structure of PR-39 in the absence or presence of lipids. According to the circular dichroic data, this structure is not altered upon incubation of PR-39 with negatively charged vesicles, although the infrared spectra suggest that the hydrogen bond pattern is modified upon the peptide-lipid interaction. This is detected by a shift in the maximum wavelength of absorption of PR-39 from 1636 cm-1 in the absence of lipids to 1645 cm-1 in the presence of lipids. We have further addressed the question of the possible mechanism of interaction of PR-39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane. PR-39 induced a calcein release from large unilamellar vesicles, which is dependent upon the peptide concentration and upon the presence of negatively charged lipid (glycerophosphoglycerol) in the membrane. The binding study of PR-39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane-bound. Addition of PR-39 to a planar lipid bilayer induced a linear increase in the current but no channel formation was observed since no discrete steps of conductance occurred.

Animals↗
PubMed1994

Generalized presence of a PEC-60-like peptide in catecholamine neurones.

PEC-60, a 60-residue intestinal peptide structurally related to the pancreatic secretory type of trypsin inhibitor, has been isolated, characterized and molecularly cloned. It shows biological activity as a hormone in both the gastrointestinal tract and in the immune system. We now report immunohistochemical evidence suggesting its neural localization exclusively within central and peripheral catecholamine (CA) neurones. PEC-60-like immunoreactivity was present in cell bodies, dendrites and nerve terminals of virtually all catecholamine neurones examined and including the noradrenergic gland cells of the adrenal medulla. PEC-60-like immunoreactivity was not seen, however, within the tyrosine hydroxylase-positive but CA-negative arcuate neurones producing growth hormone releasing hormone. The findings open up the possibility that a PEC-60-like peptide may represent a generalized co-transmitter in the peripheral and central CA neurones.

Animals↗
PubMed1994

PEC-60 increases dopamine but not GABA release in the dorsolateral neostriatum of the halothane anaesthetized rat. An in vivo microdialysis study.

The effect of striatal perfusion with the intestinal peptide PEC-60 on endogenous dopamine (DA) and gamma-aminobutyric acid (GABA) release in the dorsolateral striatum and GABA release in the globus pallidus was monitored using in vivo microdialysis in the halothane anaesthetized rat. The results show that PEC-60 (100 nM) increases DA release in the dorsolateral striatum without influencing GABA release in the dorsolateral striatum or in the globus pallidus. In addition, PEC-60 failed to influence the extracellular striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. The PEC-60 induced increase in striatal DA was abolished by the addition of tetrodotoxin (1 microM) to the perfusion medium. These data suggest that PEC-60 plays a role in modulating striatal DA release but not DA metabolism and that this effect is primarily targeted on the presynaptic DA terminals of the nigrostriatal DA pathway rather than on the postsynaptic striatopallidal GABA projection neurons in the dorsolateral striatum.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Isolation of three antibacterial peptides from pig intestine: gastric inhibitory polypeptide (7-42), diazepam-binding inhibitor (32-86) and a novel factor, peptide 3910.

Two antibacterial peptides, cecropin P1 and PR-39 (39-residue proline/arginine-rich peptide), from the upper part of pig small intestine have previously been isolated and characterized. We have now continued our search for antibacterial peptides in different side fractions generated during the isolation of intestinal hormones. Starting from one such fraction and monitoring activity against Bacillus megaterium, we isolated three homogeneous peptides by three consecutive chromatographic steps. Amino acid sequence analysis in combination with mass spectrometry identified two of the peptides as gastric inhibitory polypeptide (7-42) [GIP(7-42)] and diazepam-binding inhibitor (32-86) [DBI(32-86)], derived from factors already known. However, intact GIP and DBI have hardly any antibacterial activity by themselves. The third peptide constitutes a previously unknown structure, designated as peptide 3910 from its molecular mass. All three peptides showed good activity against B. megaterium. In addition, GIP (7-42) showed some activity against Streptococcus pyogenes and an Escherichia coli mutant with a defect in its outer membrane.

Amino Acid Sequence↗
PubMed1993

Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine.

Cecropin P1 and PR-39 are two antibacterial peptides isolated from the upper part of the small intestine of the pig. They have been sequenced, and their antibacterial spectra have been investigated (J.-Y. Lee, A. Boman, C. Sun, M. Andersson, H. Jörnvall, V. Mutt, and H. G. Boman, Proc. Natl. Acad. Sci. USA 86:9159-9162, 1989; B. Agerberth, J.-Y. Lee, T. Bergman, M. Carlquist, H. G. Boman, V. Mutt, and H. Jörnvall, Eur. J. Biochem. 202:849-854, 1991). We have now compared these two peptides for their mechanism of action on Escherichia coli K-12 by using three strains with different markers. Our results show that cecropin P1, like other cecropins, kills bacteria by lysis and that this reaction requires more peptide to kill more cells. PR-39 requires a lag period of about 8 min to penetrate the outer membrane of wild-type E. coli; then killing is quite fast. This lag period was absent in the envA1 mutant; in this strain the outer membrane was freely permeable to both peptides. PR-39 killed growing bacteria faster than nongrowing cells; for cecropin P1 there was no such difference. It is suggested from isotope incorporation experiments that PR-39 kills bacteria by a mechanism that stops protein and DNA synthesis and results in degradation of these components.

Animals↗
PubMed1992

Molecular cloning of PEC-60 and expression of its mRNA and peptide in the gastrointestinal tract and immune system.

The peptide PEC-60, structurally related to the pancreatic secretory trypsin inhibitor, inhibits glucose-induced insulin secretion. Here we report on the structure of a cDNA clone from pig duodenum encoding PEC-60. The cDNA encodes a 86-amino acid long precursor protein containing a 26-amino acid signal sequence, implying that the mature PEC-60 peptide is secreted from cells. Analysis of porcine duodenum demonstrated a high expression of a 0.6-kilobase long PEC-60 mRNA in this tissue, as well as the presence of strong PEC-60-like immunoreactivity in the cytoplasm of the majority of the goblet cells of the epithelium. High levels of PEC-60 mRNA were also found in the bone marrow and the peripheral blood and moderate levels in the spleen. A strong PEC-60-like immunoreactivity was localized in the monocytes of peripheral blood. Radioimmunoassay revealed high levels of pig PEC-60-like immunoreactivity in pig plasma suggesting that the PEC-60 peptide is efficiently released from cells. These findings imply that the gastrointestinal peptide PEC-60 is formed, stored, and secreted from monocytes present within the bone marrow and in the peripheral blood, indicating a role of the PEC-60 peptide in the immune system in addition to its function as a gastrointestinal peptide.

Amino Acid Sequence↗
PubMed1992

Protecting effects of vasoactive intestinal polypeptide on lymphocytes against metal toxicity.

The hormonal neuropeptides calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), were investigated for a potential protective effect on thymocytes after a toxic dose of nickel sulfate, giving an inhibition of DNA synthesis. There was a statistically significant increase in the synthesis of DNA from the level caused by nickel sulfate, with VIP, 10(-4)-10(-5) mol/l, while the slightly stimulating effects obtained with CGRP, CCK and NPY, were statistically non-significant. This indicates that VIP, at least as pharmacological concentrations, might have protective effects on lymphocytes against metal toxicity.

Cells, Cultured↗
PubMed1991

Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides.

We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.

Amino Acid Sequence↗
PubMed1991

Direct analysis of peptides and amino acids from capillary electrophoresis.

Capillary zone electrophoresis of peptides on a preparative scale and direct analysis of both amino acid sequences and compositions are demonstrated. Using native and synthetic peptides prepared by repetitive runs and appropriate collections, sufficient material for direct sequence analysis is obtained after just a few runs, while single runs suffice for total compositions. Electrophoretic direct amino acid analysis without derivatization is also possible and gives especially high sensitivity for aromatic residues (less than 200 fmols) plus convenient distinction of amidated versus free C-terminal residues after carboxypeptidase digestions.

Amino Acids↗
PubMed1991

Pancreatic secretory trypsin inhibitor (PSTI) isolated from pig intestine. Influence on insulin and somatostatin release.

Upon investigation of pig intestinal peptides for effects on the release of endocrine hormones from the isolated perfused rat pancreas, we reported earlier that glucose-stimulated insulin release was inhibited by PEC-60, a peptide with marked sequence similarity to PSTI (pancreatic secretory trypsin inhibitor). Continuing this study we found a polypeptide, which inhibited glucose-induced insulin release but enhanced glucose-induced somatostatin secretion. Determination of the amino acid sequence of this polypeptide revealed that it is identical to that of PSTI. Thus, PSTI modulates islet hormone release.

Amino Acids↗