FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034280103> ?p ?o ?g. }

• W2034280103 endingPage "13093" @default.
• W2034280103 startingPage "13088" @default.
• W2034280103 abstract "Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, namedCramp for cathelin-relatedantimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic α-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5–8.0 μm) and by permeabilizing the inner membrane of Escherichia colidirectly at 1 μm. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair. Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, namedCramp for cathelin-relatedantimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic α-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5–8.0 μm) and by permeabilizing the inner membrane of Escherichia colidirectly at 1 μm. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair. Endogenous antimicrobial peptides play an important role in innate immunity (1Martin E. Ganz T. Lehrer R.I. J. Leukocyte Biol. 1995; 58: 128-136Crossref PubMed Scopus (297) Google Scholar, 2White S.H. Wimley W.C. Selsted M.E. Curr. Opin. Struct. Biol. 1995; 5: 521-527Crossref PubMed Scopus (378) Google Scholar, 3Boman H.G. Cell. 1991; 65: 205-207Abstract Full Text PDF PubMed Scopus (539) Google Scholar). More than 100 microbicidal peptides have been isolated from plants and animals (4Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1520) Google Scholar). The role of these defense peptides in mammals has been inferred from their expression in neutrophil granules and at sites exposed to multiple microbes such as the skin and gastrointestinal tract. To exert their antimicrobial effect these peptides adhere to and permeabilize the surface membranes of potential pathogens. This activity is a consequence of several common features such as a high content of basic residues and the tendency of some to adopt an amphipathic conformation. However, antimicrobial peptides show marked diversity in structure and antimicrobial spectrum. One class of antimicrobial peptides, the cathelicidin-derived peptides, contains a highly conserved prepro region that is homologous to cathelin, a putative cysteine-proteinase inhibitor originally isolated from pig leukocytes (5Ritonja A. Kopitar M. Jerala R. Turk V. FEBS Lett. 1989; 255: 211-214Crossref PubMed Scopus (129) Google Scholar). Cathelicidins have been identified in several species including pig, cow, sheep, rabbit, and man (6Agerberth B. Lee J.Y. Bergman T. Carlquist M. Boman H.G. Mutt V. Jörnvall H. Eur. J. Biochem. 1991; 202: 849-854Crossref PubMed Scopus (296) Google Scholar, 7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar, 8Storici P. Zanetti M. Biochem. Biophys. Res. Commun. 1993; 196: 1058-1065Crossref PubMed Scopus (65) Google Scholar, 9Gennaro R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar, 10Bagella L. Scocchi M. Zanetti M. FEBS Lett. 1995; 376: 225-228Crossref PubMed Scopus (102) Google Scholar, 11Mahoney M.M. Lee A.Y. Brezinski-Caliguri D.J. Huttner K.M. FEBS Lett. 1995; 377: 519-522Crossref PubMed Scopus (76) Google Scholar, 12Tossi A. Scocchi M. Skerlavaj B. Gennaro R. FEBS Lett. 1994; 339: 108-112Crossref PubMed Scopus (84) Google Scholar, 13Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar). The high degree of conservation of their cathelin domain suggests that the members of this family evolved from a common ancestor gene through duplication and modification (7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar). In general, the precursors of these peptides are stored in neutrophil granules. Upon stimulation, the cathelin domain is cleaved proteolytically to allow the mature COOH-terminal antimicrobial peptide to be released. The antimicrobial portion of the cathelicidin-derived gene family is highly diverse in terms of structure and function. In the pig, the cathelicidin PR-39 has been found to have potent activity against Gram-negative bacteria and also to function as a stimulator of syndecan-1 and -4 expression on fibroblasts and endothelia (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar). Observations of functions beyond antimicrobial activity have also been made for other host defense peptides (15Murphy C.J. Foster B.A. Mannis M.J. Selsted M.E. Reid T.W. J. Cell. Physiol. 1993; 155: 408-413Crossref PubMed Scopus (187) Google Scholar) and suggest the need for further study of the role these peptides play in vivo. The mouse is a highly useful animal model to study the function of the immune system in events such as wound repair and cutaneous inflammation. Surprisingly, despite the well characterized immune system in the mouse, antimicrobial peptides have only been identified in the mouse intestine (16Yount N.Y. Wang M.-S.C. Yuan J. Banaiee N. Ouellette A.J. Selsted M.E. J. Immunol. 1995; 155: 4476-4484PubMed Google Scholar, 17Ouellette A.J. Hsieh M.W. Cano-Gauci D.F. Nosek M.T. Huttner K.M. Buick R.N. Selsted M.E. Infect. Immun. 1994; 62: 5040-5047Crossref PubMed Google Scholar) and appear to be absent from neutrophils (18Eisenhauer P.B. Lehrer R.I. Infect. Immun. 1992; 60: 3446-3447Crossref PubMed Google Scholar). Thus, in this investigation we sought to identify a mouse member of the cathelicidin gene family and describe its expression and antimicrobial function. We report a full-length cDNA sequence derived from mouse marrow which is a member of the cathelicidin gene family. This gene, named Cramp, forcathelin-relatedantimicrobial peptide, mapped to a single region on murine chromosome 9, homologous to the map locations of cathelicidins in man and pig. Transcripts for Cramp were expressed in multiple mature tissues and during embryogenesis. Finally, CRAMP protein was identified by immunostaining in murine bone marrow cells and neutrophils and behaved structurally and functionally as a potent antimicrobial agent. Amino acids and coupling reagents for peptide synthesis were from PerSeptive Biosystems (Framingham, MA) and Novabiochem (Laufelfingen, Switzerland). HPLC-grade acetonitrile, 1The abbreviations used are: HPLC, high performance liquid chromatography; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; Fmoc,N-(9-fluorenyl)methoxycarbonyl; MIC, minimum inhibitory concentration(s); PBS, phosphate-buffered saline. N-methyl-2-pyrrolidone, dichloromethane, andN,N-dimethylformamide were from Lab-Scan (Dublin, Ireland). Trifluoroacetic acid, N-methylmorpholine, and trifluoroethanol were from Janssen Chimica (Beerse, Belgium).o-Nitrophenyl-β-d-galactopyranoside was from Sigma. Mueller-Hinton broth, Bacto-agar, dextrose, mycological peptone, and yeast extract powder were from Unipath Ltd (Basingstoke, U. K.). All other chemicals were of analytical grade. Total RNA was extracted from C57BL/6 mouse femoral marrow cells with guanidinium thiocyanate (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). To isolate potential murine cDNA homologs to the cathelin-related antimicrobial peptides, a 5′ and 3′ RACE strategy was applied (Life Technologies, Inc.; 3′ and 5′ RACE systems, Gaithersburg, MD). For 3′ RACE, cDNA synthesis was carried out using adapter primer 5′-GGCCACGCGTCGACTAGTAC(T)17-3′. Amplification toward the 3′ end was done first with the universal amplification primer 5′-(CUA)4GGCCACGCGTCGACTAGTAC-3′ and with a cathelin-specific primer-1, 5′-TCGGAAGCTAATCTCTAC-3–3′, which was designed based on a base pairs 165–182 sequence of porcine prepro-PR-39. A second nested amplification was then done with a cathelin-specific primer-2, 5′-(CAU)4CTGGACCAGCCGCCCAAG-3′ designed based on base pairs 195–212 of prepro-PR-39 and the universal amplification primer. For amplification toward the 5′ end, the gene-specific primer-1, 5′-TTTGCGGAGAAGTCCAGC-3′, based on a sequence derived from 3′ RACE, was used for cDNA synthesis. Amplification was done with an anchor primer, provided by Life Technologies, Inc., and with a gene-specific primer-2, 5′-(CAU)4GAAATTTTCTTGAACCG-3′. Products of 3′ and 5′ RACE were cloned into pAMP1 for sequencing by automated sequencer (model 373A, Perkin-Elmer) in both directions using primers against M13 and T7. Three independent clones were sequenced in both directions. Northern blot analysis of total RNA was performed as described previously (14Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Crossref PubMed Scopus (328) Google Scholar). Approximately 10 μg of total RNA was extracted from whole C57BL/6 mouse embryos at different gestational ages and separated by electrophoresis through a 1% agarose/formaldehyde gel. RNA was transferred to a GeneScreen Plus membrane (DuPont NEN). Hybridization was carried out at 65 °C in QuikHyb Solution (Stratagene) and probed using [32P]dCTP random primer-labeled cDNA, corresponding to base pairs 79–249 of murine CRAMP cDNA. Filters were washed twice for 15 min in 2 × SSPE (0.18 m NaCl, 0.01 mNa2H2PO4, 1 mm EDTA, pH 7.7), 0.1% sodium dodecyl sulfate at room temperature and then twice in 0.2 × SSPE, 0.1% sodium dodecyl sulfate at 55 °C. Total RNA from marrow, testis, stomach, small intestine, liver, lung, skeletal muscle, brain, heart, spleen, kidney, and large intestine of 12-week-old C57BL/6 mice was prepared with guanidinium thiocyanate (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). Approximately 5 μg of DNase-treated total RNA from each tissue was annealed at 42 °C with random hexamers in a solution containing 10 mm Tris-HCl, pH 8.3, 50 mm KCl, and 5 mm MgCl2. The annealed hexamers were then extended by adding dNTP at a final concentration of 0.1 mm, 1.5 units of RNasin (Promega, Madison), and 1 unit of Superscript reverse transcriptase (Life Technologies, Inc.) at 42 °C for 90 min. The resulting cDNA was then amplified with the specific primers 5′-GCTGATGTCAAAAGAATCAGCG-3′ and 5′-TCCCTCTGGAACTGCATGGTTCC-3′, based on base pairs 10–32 and 357–378, respectively, of the CRAMP cDNA sequence. These primers were used with the following thermal cycle profile: 95 °C for 5 min; 20 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min; and a final extension step of 72 °C for 7 min. A second round of PCR was performed with nested primers 5′-CTGTGGCGGTCACTATCACT-3′ and 5′-GTTCCTTGAAGGCACATTGC-3′ based on base pairs 49–68 and 291–310, respectively, of the CRAMP cDNA sequence. Amplification conditions were as above, but a higher annealing temperature of 58 °C was used. Products were separated on 3% agarose gel and photographed using Eagle Eye apparatus (Stratagene). Solid phase peptide synthesis of CRAMP-1 was done on a Milligen 9050 synthesizer (Milligen, Bedford, MA). The synthesis was performed using Fmoc-l-Glu(OtBu)-PEG-PS resin (0.2mmol/g) andN,N-dimethylformamide as solvent. Couplings were carried out with a 6-fold excess of an equimolar mixture of Fmoc-amino acid, N-hydroxybenzotriazole, and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in the presence of N- methylmorpholine. Deprotection from the Fmoc group was performed with piperidine,N-methyl-2-pyrrolidone, andN,N-dimethylformamide (1:2:2, v/v) in the presence of 0.7% (v/v) 1,8-diazabicyclo (5,4,0) undec-7-ene. To improve yield, the column temperature was increased to 45 °C, the resin was washed withN-methyl-2-pyrrolidone/N,N-dimethylformamide/dichloromethane (1:1:1, v/v) containing 1% Triton X-100 and 2-methylencarbonate immediately before each coupling step, and 1-hydroxy-7-azabenzotriazole and O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate replaced N-hydroxybenzotriazole and TBTU for the coupling of residues 7–11, 15–18, and 24. CRAMP-2 was obtained by elongation of CRAMP-1. Amino acid side chains were protected with trityl (Gln, Asn), t-butyloxycarbonyl (Lys),t-butyl (Glu, Ser), and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg). Peptide deprotection and cleavage from the resin were carried out using a mixture of trifluoroacetic acid/ethandithiol/phenol/water/triisopropylsilane (90:4:2:2:2, v/v) for 2 h at room temperature. After cleavage, both peptides were extracted repeatedly with ethyl ether and purified by reverse phase HPLC on a C18 Delta-Pak column (Waters, Bedford, MA), using a 0–60% water/acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The peptide concentration was measured by the absorbance of Phe at 257 nm and using a molar extinction coefficient of 195 (20Buck M.A. Olah T.A. Weitzmann C.J. Cooperman B.S. Anal. Biochem. 1989; 182: 295-299Crossref PubMed Scopus (66) Google Scholar). Mass determinations were performed with an API I ionspray mass spectrometer (PE SCIEX, Toronto, Canada). Circular dichroism spectra were recorded at room temperature on a Jasco J-600 spectropolarimeter (Jasco Corp., Tokyo, Japan). Peptide samples (10–25 μm) were dissolved in 5 mm sodium phosphate buffer, pH 7.0, in the absence or presence of 15, 30, and 45% (v/v) trifluoroethanol. The α-helical content was estimated by the method of Chen et al. (21Chen Y.-H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1965) Google Scholar) from the [θ]222measurements, using −1,500 and −39,500 (1–2.5/n) deg-cm2 dmol−1, as the values for 0% and 100% of helix, respectively, with n indicating the number of residues. Antibacterial and antifungal minimum inhibitory concentrations (MIC) were determined by a microdilution susceptibility test described previously (9Gennaro R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar). The following bacterial strains were tested: Escherichia coli ATCC 25922, ML-35 and D21,Salmonella typhimurium ATCC 14028, Pseuodomonas aeruginosa ATCC 27853, Serratia marcescens ATCC 1800,Proteus vulgaris ATCC 13315, Staphylococcus aureus ATCC 25923, Cowan 1, and two methicillin-resistant clinical isolates carrying the mecA gene (provided by L. Dolzani, Department of Biomedical Sciences, University of Trieste, Italy),Staphylococcus epidermidis ATCC 12228, and Bacillus megaterium BM 11. The MIC values were determined after incubation at 37 °C for 18 h. The antifungal activity was determined using clinical isolates of Candida albicans and Cryptococcus neoformans. The assay conditions were similar to those used for bacteria, except that the fungal species were grown and tested in Sabouraud liquid medium, and the MIC was determined after incubation at 30 °C for 36–48 h. The bactericidal activity of CRAMP-1 and CRAMP-2 against midlog phase cultures of E. coli ATCC 25922, P. aeruginosaATCC 27853, and S. aureus ATCC 25923 was tested in low ionic strength buffer (10 mm sodium phosphate buffer, pH 7.4). Bacteria (0.4–0.6 × 106 colony-forming units/ml) were incubated in the absence (controls) or in the presence of different amounts of peptides in a 96-well microtiter plate (final volume of 150 μl). After a 1-h incubation at 37 °C, samples were serially diluted with sterile PBS, plated in duplicate on Mueller-Hinton agar, and incubated for 16–18 h to allow colony counts (9Gennaro R. Skerlavaj B. Romeo D. Infect. Immun. 1989; 57: 3142-3146Crossref PubMed Google Scholar). Bacterial inner membrane permeabilization was evaluated with theE. coli ML-35 strain as described previously (22Skerlavaj B. Romeo D. Gennaro R. Infect. Immun. 1990; 58: 3724-3730Crossref PubMed Google Scholar). Antibodies to synthetic CRAMP-1 were raised in New Zealand White rabbits by an initial intramuscular injection of 150 μg of CRAMP-1 in complete Freund's adjuvant followed by five booster injections of 150 μg of CRAMP in incomplete Freund's at 3-week intervals. Serum was collected 10 days after boosting. 4 × 105Swiss-Webster mouse bone marrow cells in 100 μl of PBS, 1% bovine serum albumin, or 5 × 106 C57BL/six peripheral blood cells in Tris-buffered saline, 5 mm EDTA, were attached to glass slides by cytospin at 400 × g for 5 min in a Shandon Cytospin 3 cytocentrifuge, fixed in methanol, then blocked for 1 h with a solution of 5% goat serum in PBS, 1% bovine serum albumin. Slides were then incubated for 1 h with a 1:200 dilution of rabbit anti-CRAMP antiserum or nonimmune rabbit serum. To confirm specificity of antibody binding, parallel slides were treated identically with rabbit-anti CRAMP serum that had been preincubated for 1 h at room temperature with 20 μg/ml synthetic CRAMP-1. Following primary antibody incubation slides were washed three times for 5 min in PBS and then incubated for 1 h at room temperature with a 1:250 dilution of fluorescein-conjugated goat anti-rabbit IgG (Cappel Research Products, Durham, NC). Each slide was washed three times for 5 min with Tris-buffered saline. Coverslips were mounted with Prolong 228 Antifade mounting medium (Molecular Probes, Eugene, OR) and cells photographed with Kodak Elite II Ektachrome ASA400 film on a Zeiss Axiophot microscope. Cramp was mapped by analysis of the progeny of two multilocus crosses: (NFS/N or C58/J × Mus m. musculus) × M. m. musculus (23Kozak C.A. Peyser M. Krall M. Mariano T.M. Kumar C.S. Pestka S. Mock B.A. Genomics. 1990; 8: 727-731Crossref PubMed Scopus (128) Google Scholar) and (NFS/N ×M. spretus) × M. spretus or C58/J (24Adamson M.C. Silver J. Kozak C.A. Virology. 1991; 183: 778-781Crossref PubMed Scopus (134) Google Scholar). Progeny of these crosses have been typed for over 1000 markers including the Chr 9 markers TRF (transferrin), Gnatl (guanine nucleotide-binding protein, α transducing subunit), Scn10a(peripheral sodium channel 3, subunit 10), and Cck(cholecystokinin) as described previously (25Ibaraki K. Kozak C.A. Wewer U.M. Albrechtsen R. Young M.F. Mamm. Genome. 1995; 6: 693-696Crossref PubMed Scopus (17) Google Scholar, 26Kozak C.A. Sangameswaran L. Mamm. Genome. 1996; 7: 787-788Crossref PubMed Scopus (17) Google Scholar). Data were stored an analyzed using the program LOCUS developed by C. E. Buckler (NIAID, Bethesda, MD). Recombinational distances and S.E. were calculated according to Ref. 27Green E.L. Genetics and Probability in Animal Breeding Experiments. Macmillan, New York1981Crossref Google Scholar. Genes were ordered by minimizing the number of recombinants. To isolate potential murine homologs to the family of antimicrobial peptides related by a cathelin-like domain, primers were designed using sequence information from the conserved cathelin-like domain of the porcine gene PR-39. A nested RACE PCR strategy was used, capitalizing on the highly invariant cathelin-like prepro domain from all species studied to date. Three independently derived clones were sequenced in both directions to give the cDNA sequence shown in Fig. 1. No other related cDNAs were identified by this approach despite screening more than 500 clones for isolates related by the 5′ cathelin domain but distinct in the 3′ region. The murine cathelin-related cDNA contains an open reading frame of 516 base pairs and encodes a predicted translation product of 172 amino acids. Sequence comparisons between the prepro regions of the predicted murine (CRAMP) pig (PR-39) and human (FALL-39) cathelicidins identify significant primary sequence similarities; the murine protein maintains 52% identity with PR-39 and FALL-39 in the cathelin-like domain and 80% identity with either individually (Fig. 2). This sequence similarity includes the conservation of cysteines at positions 83, 94, 105, 122 and potential processing sites at the carboxyl terminus of the cathelin portion of the peptide. The previously reported cDNA sequences of human FALL-39 and porcine PR-39 encode different peptides in the 3′ region. Similarly, the predicted translation product of CRAMP is distinct in this region. To evaluate the expression of Cramp mRNA in the mouse, RNA was extracted from whole murine embryos isolated at various gestational ages and from whole adult mouse tissues. Northern blot of total RNA from whole embryos demonstrated abundant 1-kilobase message (Fig. 3, A and B) mRNA was detectable as early as gestational days 12 and 13 and increased relative to ribosomal RNA during development. In adult tissues, this transcript was detectable by Northern blot analysis only in marrow total RNA (data not shown). To increase the sensitivity for detection in adult tissues, a reverse transcriptase-PCR strategy was employed. Total RNA from brain, heart, spleen, kidney, testis, colon, liver, marrow, stomach, small intestine, lung, and skeletal muscle was prepared, and reverse transcriptase-PCR was performed using nested primers to specifically amplify cDNA. As a control for all reverse transcriptase-PCR experiments, primers selected for β-actin were chosen, and reactions were included lacking either RNA or reverse transcriptase. Cramp transcripts were detectable in spleen, testis, colon, marrow, stomach, and small intestine (Fig.3 C). Faint bands corresponding to Cramp were also seen in heart, lung, and skeletal muscle but were not detectable in brain, kidney, and liver. All samples were positive with actin-specific primers and negative when performed in the absence of RNA or reverse transcriptase. Using the probe spanning base pairs 79–249 of Cramp, we identifiedScaI fragments of 10.2 kilobases in parental M. musculus DNA, 11.7 in NFS/N and C58/J, and 14.2 in M. spretus. Inheritance of the variant fragments was typed in the progeny of two sets of genetic crosses and compared with inheritance of more than 1000 genetic markers previously typed and mapped in these mice. CRAMP showed linkage to markers on distal Chr 9 (Fig.4) and was positioned just distal to Gnat1. This region of mouse Chr 9 is homologous to human 3p23-21, consistent with the location of the human homolog of this gene,Fall-39. The translation products of the cathelicidins are processed to release carboxyl-terminal peptides with antimicrobial activity. To investigate the properties of the carboxyl-terminal peptide predicted by the CRAMP cDNA, two peptides were synthesized. The first peptide, CRAMP-1, was prepared based on the predicted elastase cleavage site at position 138–139 and is a 33-amino acid peptide. The second, CRAMP-2, was prepared based on potential processing at the dibasic site at position 133–134 and is a 38-amino acid peptide. Helical wheel projections of both CRAMP-1 and CRAMP-2 predict that these peptides will form an amphipathic helix similar to that for FALL-39 (13Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (435) Google Scholar) and non-cathelin-related antimicrobial peptides such as the maganins and cecropins. To evaluate this directly, circular dichroism (CD) spectral analysis of CRAMP-1 and CRAMP-2 was done (Fig. 5). The spectra of 10–25 μm synthetic CRAMP-1 in buffer suggest that the peptide is in a random coil configuration and that it will assume a helical structure in the presence of 15–45% (v/v) trifluoroethanol. The CD spectra of CRAMP-2 (not shown) were similar to CRAMP-1. The capacity of CRAMP-1 and CRAMP-2 to function as antimicrobial agents was evaluated directly on a variety of microbes (Table I). Both peptides demonstrated potent antimicrobial activity against Gram-negative bacteria with MIC values in the range of 0.5–8.0 μm. CRAMP peptides were less active against Gram-positive strains (MIC 32–64 μm) and not active at the concentrations tested against P. vulgaris, C. albicans, and selected strains of S. aureus. The CRAMP peptides were also found to have potent direct bactericidal activity when assayed in low ionic strength buffer (Table II). Under these conditions even the Gram-positive bacterium (S. aureus) was killed at concentrations as low as 1 μm.Table IAntimicrobial activity of CRAMP-1 and CRAMP-2Organism and strainMICCRAMP-1CRAMP-2μmEscherichia coli ATCC 2592211Escherichia coli ML3522Escherichia coli D210.50.5Salmonella typhimurium ATCC 1402888Pseudomonas aeruginosa ATCC 2785344Serratia marcescens ATCC 810044Proteus vulgaris ATCC 13315>64>64Staphylococcus aureus ATCC 259233232Staphylococcus aureus Cowan I3232Staphylococcus aureus (MRSA)>64>64Staphylococcus aureus (MRSA)6464Staphylococcus epidermidis ATCC 122281616Streptococcus faecalis ATCC 292123216Bacillus megaterium Bm1144Candida albicans>64>64Cryptococcus neoformans1616MIC was defined as the lowest concentration of peptide preventing visible growth after incubation with bacteria for 18 h or with fungi for 36–48 h. All strains tested were grown in Mueller-Hinton broth, except B. megaterium, which was grown in LB medium, and C. albicans and C. neoformans, which were grown in Sabouraud liquid medium. Results were determined using approximately 1.5 × 105 (bacteria) and 0.5 × 105 (fungi) colony-forming units/ml and are the mean of at least three independent determinations with a divergence of not more than one MIC value with respect to those reported here. Open table in a new tab Table IIBactericidal activity of CRAMP-1 and CRAMP-2ConcentrationE. coli ATCC 25922S. aureus ATCC 25923P. aeruginosa ATCC 27853μm%killed bacteriaCRAMP-10.118.95.8120.361 –65.432.7 –40.4421.073 –99.191.2 –>99.9995.510.0>99.99 –>99.99>99.99 –>99.99>99.99CRAMP-20.381.6 –57.893.7 –89.948.61.097.1 –99.599.8 –>99.9999.810.0>99.99 –>99.99>99.99 –>99.99>99.99The microbicidal activity of CRAMP-1 and CRAMP-2 was assessed by incubating midlog phase bacteria (0.4–0.6 × 106colony-forming units/ml) with the indicated amount of peptide in the presence of 10 mm sodium phosphate buffer, pH 7.4 (final volume of 150 μl). After a 1-h incubation at 37 °C, samples were serially diluted with sterile PBS, plated in duplicate on Mueller-Hinton agar, and incubated for 16–18 h to allow colony counts. Results are expressed as percent of killed bacteria with respect to controls incubated in the absence of peptide. Open table in a new tab MIC was defined as the lowest concentration of peptide preventing visible growth after incubation with bacteria for 18 h or with fungi for 36–48 h. All strains tested were grown in Mueller-Hinton broth, except B. megaterium, which was grown in LB medium, and C. albicans and C. neoformans, which were grown in Sabouraud l" @default.
• W2034280103 created "2016-06-24" @default.
• W2034280103 creator A5001064163 @default.
• W2034280103 creator A5009913321 @default.
• W2034280103 creator A5034224042 @default.
• W2034280103 creator A5042304008 @default.
• W2034280103 creator A5051994090 @default.
• W2034280103 creator A5068876318 @default.
• W2034280103 creator A5070070418 @default.
• W2034280103 date "1997-05-01" @default.
• W2034280103 modified "2023-10-06" @default.
• W2034280103 title "Identification of CRAMP, a Cathelin-related Antimicrobial Peptide Expressed in the Embryonic and Adult Mouse" @default.
• W2034280103 cites W1481684176 @default.
• W2034280103 cites W1522262409 @default.
• W2034280103 cites W1836919226 @default.
• W2034280103 cites W1896164923 @default.
• W2034280103 cites W1902412925 @default.
• W2034280103 cites W1951818427 @default.
• W2034280103 cites W1969147661 @default.
• W2034280103 cites W1970488666 @default.
• W2034280103 cites W1981854518 @default.
• W2034280103 cites W1985881522 @default.
• W2034280103 cites W1989452309 @default.
• W2034280103 cites W2000513200 @default.
• W2034280103 cites W2001959707 @default.
• W2034280103 cites W2002193923 @default.
• W2034280103 cites W2011322938 @default.
• W2034280103 cites W2021413161 @default.
• W2034280103 cites W2031299124 @default.
• W2034280103 cites W2037701623 @default.
• W2034280103 cites W2038717313 @default.
• W2034280103 cites W2050242176 @default.
• W2034280103 cites W2051142131 @default.
• W2034280103 cites W2057338838 @default.
• W2034280103 cites W2061413267 @default.
• W2034280103 cites W2066432956 @default.
• W2034280103 cites W2083845392 @default.
• W2034280103 cites W2084574358 @default.
• W2034280103 cites W2085269931 @default.
• W2034280103 cites W2094547651 @default.
• W2034280103 cites W2126516803 @default.
• W2034280103 cites W2128112850 @default.
• W2034280103 cites W2130974803 @default.
• W2034280103 cites W2138726430 @default.
• W2034280103 cites W2144599944 @default.
• W2034280103 cites W2163168560 @default.
• W2034280103 cites W2171433457 @default.
• W2034280103 cites W4294216491 @default.
• W2034280103 doi "https://doi.org/10.1074/jbc.272.20.13088" @default.
• W2034280103 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9148921" @default.
• W2034280103 hasPublicationYear "1997" @default.
• W2034280103 type Work @default.
• W2034280103 sameAs 2034280103 @default.
• W2034280103 citedByCount "375" @default.
• W2034280103 countsByYear W20342801032012 @default.
• W2034280103 countsByYear W20342801032013 @default.
• W2034280103 countsByYear W20342801032014 @default.
• W2034280103 countsByYear W20342801032015 @default.
• W2034280103 countsByYear W20342801032016 @default.
• W2034280103 countsByYear W20342801032017 @default.
• W2034280103 countsByYear W20342801032018 @default.
• W2034280103 countsByYear W20342801032019 @default.
• W2034280103 countsByYear W20342801032020 @default.
• W2034280103 countsByYear W20342801032021 @default.
• W2034280103 countsByYear W20342801032022 @default.
• W2034280103 countsByYear W20342801032023 @default.
• W2034280103 crossrefType "journal-article" @default.
• W2034280103 hasAuthorship W2034280103A5001064163 @default.
• W2034280103 hasAuthorship W2034280103A5009913321 @default.
• W2034280103 hasAuthorship W2034280103A5034224042 @default.
• W2034280103 hasAuthorship W2034280103A5042304008 @default.
• W2034280103 hasAuthorship W2034280103A5051994090 @default.
• W2034280103 hasAuthorship W2034280103A5068876318 @default.
• W2034280103 hasAuthorship W2034280103A5070070418 @default.
• W2034280103 hasBestOaLocation W20342801031 @default.
• W2034280103 hasConcept C104317684 @default.
• W2034280103 hasConcept C116834253 @default.
• W2034280103 hasConcept C145103041 @default.
• W2034280103 hasConcept C2779281246 @default.
• W2034280103 hasConcept C4937899 @default.
• W2034280103 hasConcept C55493867 @default.
• W2034280103 hasConcept C59822182 @default.
• W2034280103 hasConcept C86803240 @default.
• W2034280103 hasConcept C89423630 @default.
• W2034280103 hasConceptScore W2034280103C104317684 @default.
• W2034280103 hasConceptScore W2034280103C116834253 @default.
• W2034280103 hasConceptScore W2034280103C145103041 @default.
• W2034280103 hasConceptScore W2034280103C2779281246 @default.
• W2034280103 hasConceptScore W2034280103C4937899 @default.
• W2034280103 hasConceptScore W2034280103C55493867 @default.
• W2034280103 hasConceptScore W2034280103C59822182 @default.
• W2034280103 hasConceptScore W2034280103C86803240 @default.
• W2034280103 hasConceptScore W2034280103C89423630 @default.
• W2034280103 hasIssue "20" @default.
• W2034280103 hasLocation W20342801031 @default.
• W2034280103 hasOpenAccess W2034280103 @default.
• W2034280103 hasPrimaryLocation W20342801031 @default.
• W2034280103 hasRelatedWork W2020469237 @default.

• next