Previous Article | Next Article 
Infect Immun, March 1998, p. 1225-1232, Vol. 66, No. 3
Department of Molecular and Cellular
Engineering, The Institute for Human Gene Therapy, The University of
Pennsylvania Medical Center, and The Wistar Institute, Philadelphia,
Pennsylvania 19104-4268
Received 22 August 1997/Returned for modification 29 October
1997/Accepted 2 December 1997
One component of host defense at mucosal surfaces appears to be
epithelium-derived peptides with antimicrobial activity called defensins. Human Antimicrobial peptides have been
found in a wide array of animal species, ranging from insects to lower
vertebrates and mammals (8). These peptides contribute to
innate host defense against a number of bacterial and fungal pathogens
(11). Mammalian defensins are small antimicrobial peptides
(3.5 to 4.5 kDa) that are characterized by the presence of six
cysteines which form three disulfide bonds whose ordered array defines
the classification as
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mouse
-Defensin 1 Is a Salt-Sensitive
Antimicrobial Peptide Present in Epithelia of the Lung and
Urogenital Tract
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-defensin 1 (hBD-1) represents the first member of
the -defensin family isolated from humans and has been implicated in
the pathogenesis of cystic fibrosis. We describe in this report the
isolation and characterization of a murine homolog of hBD-1 called
mouse -defensin 1 (mBD-1). The predicted amino acid sequence shows
the hallmark features of other known epithelial -defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-1 revealed two exons separated by a 15-kb
intron. By use of fluorescence in situ hybridization, the mBD-1 gene
was localized at the proximal portion of chromosome 8, the site where
mouse 
-defensins are found. Lysates from cells transfected with the
mBD-1 cDNA showed antibacterial activity against gram-positive and
gram-negative bacteria. mBD-1 transcripts were found in kidney, liver,
and female reproductive organ tissues. In the airways, mBD-1 is
expressed diffusely throughout the epithelial cells of the large
proximal airways with less expression in the small distal airways and
no expression in alveolar cells. The present study demonstrates that a
-defensin potentially homologous to human -defensin 1 is present
in the respiratory system and other mucosal surfaces in mice.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
- or -defensins. The -defensins are
found in granulocytes (8) and Paneth cells of the small
intestine (14, 18) in various species, whereas -defensins
occur in leukocytes of cattle (28, 31) and fowl (7,
12). Further, -defensins are expressed in epithelial cells,
where they contribute to the host defense system of mucosal surfaces.
Lingual and tracheal antimicrobial peptides are -defensins expressed
in bovine epithelial cells of the tongue and trachea (2, 4,
26). The first -defensin isolated from humans, human
-defensin 1 (hBD-1), is found in salivary gland, airway, prostate,
and placenta tissues, among other tissues (1, 10, 19,
32).
View larger version (35K):
[in a new window]
FIG. 1.
cDNA and peptide sequences of mBD-1. (A) cDNA and
deduced amino acid sequences of mBD-1. The first underline indicates
the putative mature peptide; the dash represents the termination codon;
the second underline indicates the polyadenylation signal. (B)
Comparison of the putative prepropeptide sequences of mBD-1, hBD-1,
TAP, and LAP, as well as the peptide sequences of bovine neutrophil
-defensins 1 and 11 (BNBD-1 and BNBD-11, respectively) and
gallinacin 1 (GAL-1). The bottom line presents the consensus sequence
of -defensins.
It has been suggested that the salt-sensitive defect of antimicrobial
peptides may underlie the pathogenesis of cystic fibrosis (CF) lung
disease (10, 29). Smith et al. (29) proposed that the defect in the CF gene product, the CF transmembrane conductance regulator, leads to elevated NaCl concentrations in the airway surface
fluid, which inactivates the antimicrobial activity, leading to
colonization of the airway with bacterial pathogens, which is the
hallmark of the disease (9, 10, 15, 28, 29). Our data
suggests that hBD-1 is one of the antimicrobial molecules disabled at
the airway surface in CF (10). It would be of great importance to have a suitable animal model, such as a mouse or rat
model, to study the role of -defensins in pulmonary host defense,
although little is known about defensin biology in rodents. Cryptins, which are expressed in the Paneth cells of the small intestine, are the only defensins identified in the mouse (5, 13,
22, 23); notably, mouse neutrophils lack defensins
(6).
The aim of the present study was to evaluate defensin activity in
epithelia of the mouse. We isolated a murine -defensin, designated mouse -defensin 1 (mBD-1), and analyzed its gene
structure, antimicrobial function, and expression patterns.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Cloning of mBD-1 cDNA and genomic sequence.
The hBD-1 cDNA sequence (10) was used to perform a BLAST
search at the website of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). A 444-bp mouse cDNA
expressed sequence tag submitted by Marra et al. (accession no.
AA065510) was found that showed 51% identity with hBD-1 at the amino
acid level, including the six -defensin-specific cysteines. Reverse
transcriptase (RT)-PCR was used to clone the full-length cDNA sequence.
Total RNA was isolated from a C57BL/6 mouse kidney by using Trizol
(Gibco BRL) and further purified to poly(A)+ RNA by using
oligo(dT) columns (Qiagen). Poly(A)+ RNA (approximately 100 ng) was reverse transcribed by using NotI-(dT)18 as the
primer (First-Strand cDNA Synthesis Kit; Pharmacia Biotech), and 10%
of the reaction product was used for a PCR. Primers specific to mBD-1
were designed as follows: forward primer (m-def 1),
5'-CGAAGCTTCACATCCTCTCTGCACTCTGG-3' (nucleotides 4 to 24 of
the GenBank sequence plus nine nucleotides at the 5' end containing a
restriction site for HindIII); reverse primer (m-def 2),
5'-CGACTAGTCCAGGCAGATGTTCTGG-3' (nucleotides 433 to 444 of
the GenBank sequence plus eight nucleotides at the 5' end
containing a restriction site for SpeI). The PCR products were analyzed on a 1.5% agarose gel, and a band of 440 bp was isolated
and subcloned into Bluescript II SK
(Stratagene).
Cloning of the genomic sequence.
A mouse
genomic library constructed in vector FIX II (Stratagene) was
screened by standard procedures (25) with a probe generated
by PCR using mBD-1 cDNA as the template and gene-specific primers
(forward, 5'-TTTCACATCCTCTCTGCACT-3' or
5'-TGCACTCTGGACCCTGGCT-3'; reverse,
5'-ACCTGGCTCCATCTGGGAGA-3' or
5'-CCATCTGGGAGAAAAGAAAACA-3'). Positive clones were
purified, and genomic DNA was isolated and subcloned into
Bluescript II KS. The genomic clones were
analyzed by partial sequencing and digestion with restriction
endonucleases.
Analysis of the chromosomal localization by fluorescence in situ hybridization (FISH). Two gene-specific primers were used to amplify a sequence spanning a region including parts of exon 1 and the intron. The PCR product was used as a probe to screen a mouse genomic bacterial artificial chromosome library. The DNA of positive clones was labeled with digoxigenin dUTP by nick translation and hybridized to normal mouse metaphase chromosomes. The chromosome of interest was identified by analysis of the 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) banding pattern and finally by cohybridization of the mBD-1-specific probe with a probe specific for the telomeric region of chromosome 8 (GenomeSystems, Inc.).
Testing of antimicrobial activity.
For testing of
antibacterial activity, we used lysates from cells transfected with the
mBD-1 cDNA in a transfection vector. Staphylococcus aureus
(clinical isolate provided by the Microbiology Laboratory, University
of Pennsylvania Health System), Escherichia coli D31
(30), and Pseudomonas aeruginosa (clinical
isolate provided by the Microbiology Laboratory, University of
Pennsylvania Health System) were used as test organisms in both
diffusion assays and liquid broth assays. To obtain the cell lysates,
human SW 13 (ATCC CCL 105) adrenal adenocarcinoma cells grown in
Dulbecco modified Eagle medium with 2.5% fetal calf serum in the
absence of antibiotics were transfected with pcDNA3.1
(Invitrogen) containing the full-length mBD-1 cDNA by using calcium phosphate methods (Profection Mammalian Transfection Systems; Promega).
As a control, we used cells transfected with the vector containing no
insert. Transfected cells and mock-transfected control cells were
harvested 48 h posttransfection, pelleted by centrifugation, resuspended in 500 µl of distilled water, and lysed by brief
sonication.
-defensins (27). Bacteria (5 × 104 CFU) grown in Luria-Bertani broth were exposed at
mid-log phase to 50 µl of the cell lysates for 2 h at 37°C.
Cell viability was measured by plating several dilutions and counting
colonies the following day. Antibacterial diffusion assays were
performed as described by Lehrer et al. (16), with some
modifications. Bacteria were grown to mid-log phase in a solution of
yeast extract (5 g/liter) and tryptone (10 g/liter) buffered with 10 mM
piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES)
at pH 7.5, and 5 × 106 CFU were diluted in 10 ml of a
solution additionally containing 1% agarose. After being poured onto
150-mm-diameter plates and cooling, cell lysates (5 µl) were pipetted
into wells formed with a 4-mm cork corer and incubated at 30°C
overnight. To analyze the salt dependency of the activity of mBD-1
against E. coli D31, NaCl was added to the agarose solution
to obtain final NaCl concentrations of 50, 100, 200, and 500 mM. The
exact NaCl concentrations in the solutions were measured by Na- or
Cl-sensitive electrodes (Microelectrodes). The pH dependency of mBD-1
activity was studied by adjusting the pH of the agarose solution
to 9.0 or 5.5 by adding NaOH or HCl. As a positive control of
antibacterial activity, 5 µl of magainin 1 (Magainin Pharmaceuticals,
Inc.) at 1 mg/ml was applied to agarose plates. Further, liquid broth
assays were performed by adding NaCl (final concentrations, 7, 50, 100, 200, and 500 mM) to the cell lysates.
RNase protection assays.
RNase protection assays were used
to measure the level of mBD-1 transcripts in lung tissue. Total RNA
from lung-trachea, skeletal muscle, and kidney tissues was isolated as
described above. [32P]dCTP-labeled antisense riboprobes
were prepared by in vitro transcription from the T7 RNA polymerase
promoters of linearized Bluescript KS II containing mBD-1
(304 bp) or -actin cDNA (265 bp) (SP6/T7 Transcription Kit,
Boehringer Mannheim). Hybridizations were performed in separate tubes
by using 5 × 105 cpm of either an hBD-1 or a
-actin probe and 20 µg of total RNA. RNA-RNA hybrids were digested
with RNases A and T1 to yield protected fragments of 261 and 237 bp for mBD-1 and -actin, respectively (Lysate Ribonuclease
Protection Kit; Amersham). Results were visualized by running the
reaction products on a urea-polyacrylamide gel, followed by
autoradiography.
RT-PCR.
A nested RT-PCR was used to detect low levels of
mBD-1 mRNA in several tissues. Poly(A)+ RNA was isolated
from mouse trachea, lung, tongue, esophagus, small bowel, large bowel,
gall bladder, pancreas, skeletal muscle, heart, fallopian tube, ovary,
vagina, and brain tissues and reverse transcribed as described above.
The following primers were used in the first round of PCR:
forward primer (m-def 5), 5'-TGGACCCTGGCTGCCACCACTATG-3'; reverse primer (m-def 6), 5'-GCTCATTCTTCAAACTACTGTCAG-3'.
The products of this PCR were diluted 1:50 in distilled
water, and an aliquot (2 µl) was used as the template for a nested
PCR using the same reaction conditions as for the first round with the
following primers: forward primer (m-def 7),
5'-ATGAAAACTCATTACTTTCTCCTGGTGATG-3'; reverse primer (m-def
8), 5'-CAATCCATCGCTCGCCTTTATGCTC-3'. The predicted size of
the PCR product was 252 bp. RT was omitted from the negative control,
whereas an RT-PCR with primers specific for mouse -actin was used as
a positive control. The PCR products were analyzed on a 1.5% agarose
gel.
In situ hybridization.
Adult mouse nose, trachea, lung,
kidney, liver, tongue, and heart tissues were harvested from
CO2-euthanized animals, embedded in OCT (Tissue-TCK; Miles
Inc.), and cryosectioned. Tissue sections 6 µm thick were mounted on
slides and fixed in 4% paraformaldehyde in phosphate-buffered saline
(pH 7.4) for 4 h at 4°C. Following dehydration through graded
concentrations of ethanol, sections were desiccated overnight under
vacuum and stored at 
20°C. The following day, sections were treated
with 10-µg/ml proteinase K for 30 min at 30°C, rinsed twice in
0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 sodium citrate) for
30 s each time, fixed in 4% paraformaldehyde in
phosphate-buffered saline, rinsed twice in 0.1 M triethanolamine (pH
8.0) for 4 min each time, incubated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min at room temperature, and dehydrated
through ethanol. Following drying under vacuum, prehybridization was
performed for 4 h at 54°C in 10 mM Tris (pH 8.0)-50%
formamide-2.5× Denhardt's solution-0.6 M NaCl-1 mM EDTA-0.1%
sodium dodecyl sulfate-500 µg of tRNA per ml-10 mM dithiothreitol
(DTT). RNase control sections were treated with 200 µg of RNase A per
ml for 1 h at 37°C before the prehybridization step. Sections
were then hybridized with 35S-labeled antisense or sense
probes at 5 × 106 cpm/ml for 16 h at 54°C in
the prehybridization solution. Probes were synthesized by in vitro
transcription from the T7 or T3 RNA polymerase promoter of full-length
mBD-1 cDNA cloned in Bluescript KS II (Promega In Vitro
Transcription System). Following prehybridization, slides were washed
in 4× SSC for 20 min at room temperature, treated with RNase (20 µg/ml) for 30 min at 37°C, and washed once with 2× SSC-1 mM DTT
at room temperature for 10 min and three times in 0.5× SSC-1 mM DTT
at 54°C. Slides were dehydrated with ethanol and air dried
before dipping in photoemulsion (Kodak). Slides were developed
and analyzed by bright- and dark-field microscopy using a Microphot-FXA
Nikon microscope.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Cloning of the cDNA and genomic sequence for mBD-1
and localization of the mBD-1 gene.
An expressed sequence
tag homologous to the cDNA sequence of hBD-1 was obtained by performing
a BLAST search at the NCBI website (Fig. 1A). This cDNA clone showed
62% nucleotide identity with the cDNA of hBD-1; the encoded peptide
was 51% identical to the hBD-1 peptide. The murine clone was called
mBD-1. The predicted peptide of mBD-1 showed the presence of the
-defensin-specific conserved amino acids, including the typical
array of six cysteines (Fig. 1B). A cDNA clone for mBD-1 was isolated
from kidney RNA by RT-PCR. A single product of the predicted size (440 bp) was obtained and cloned into Bluescript II SK. The
insert was sequenced and found to be identical to the database clone
(Fig. 1A). The mBD-1 cDNA sequence consists of a 204-bp open reading
frame encoding a peptide of 68 amino acids with a putative prepro
sequence (Fig. 1A and B).
-defensin-related mouse or human sequences,
although a cDNA clone (accession no. X89820) obtained from a rat was found to be 94% identical to the mBD-1 cDNA.
|
|
Analysis of antimicrobial activity. To test whether the cloned cDNA codes for an antimicrobial peptide, lysates from cells transfected with the mBD-1 cDNA were used in different assays and found to be active against all of the bacteria used, as well as in the diffusion assay (data not shown) and the liquid broth assay (Fig. 4A to C). Lysates from cells transfected with an empty vector failed to demonstrate killing activity, supporting the specificity of the assay (Fig. 4A to C). These results indicate that mBD-1 has antimicrobial activities against both gram-positive and gram-negative bacteria. Further, this activity is substantially diminished at high concentrations of NaCl (Fig. 4D) and at an acidic pH (data not shown).
|
Expression of mBD-1 in mouse tissues.
RNA from various tissues
was also evaluated for mBD-1 transcripts by RNase protection analysis.
This sensitive assay detected mBD-1 mRNA from an extract of the whole
lung (i.e., the lung and trachea) but at much lower levels than in the
kidney. This assay failed to detect mBD-1 RNA in many other tissues,
such as muscle (Fig. 5A). A
-actin probe was used as an internal control.
|
-Actin was again used
as a positive control. No bands were detected when RT was omitted (data
not shown).
The tissue distribution of mBD-1 was analyzed at the cellular level by
in situ hybridization. mBD-1 transcripts were detected in the epithelia
of the nose (Fig. 6A and B), the large
cartilaginous airways (Fig. 6E and F), and the larger bronchioles
(Fig. 6I and J) when tissue sections were hybridized to the
antisense probe. Hybridization signal appeared diffusely throughout
virtually all of the surface epithelial cells of the conducting airway.
The signal was substantially reduced or absent in the small bronchioles or lung parenchyma (Fig. 6M and N). Positive results were also seen in
the distal tubules and collecting ducts of the kidney (Fig. 7A and
B) and the epithelium covering the tongue
(Fig. 7E and F) and as a diffuse signal in the liver (Fig. 7I-J). No
signal over the background was detected in the heart muscle (Fig. 7M and N). Hybridization with sense mBD-1 riboprobes (Fig. 6 and 7) or
digestion of the tissue with RNase before hybridization to the
antisense probe (data not shown) resulted in no detectable signal,
confirming the sensitivity of the assay.
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In the present report, we describe the isolation of a mouse cDNA with homology to hBD-1 whose expressed peptide in transfected cells has antimicrobial activity against various gram-positive and gram-negative organisms. We have named this antimicrobial peptide mBD-1.
The cDNA sequence was found during a BLAST search using the nucleotide
sequence of hBD-1. Whereas the similarity of mBD-1 to hBD-1 is only
62% on the nucleotide level and 51% on the amino acid level, the
amino acid sequence of the putative mBD-1 prepropeptide revealed the
structural hallmarks of -defensins. The amino-terminal prepro
portion of the peptide contains several hydrophobic residues which are
characteristic of -defensins and are found in other -defensin
family members expressed on mucosal surfaces, such as the tracheal
antimicrobial peptide (TAP) (2, 4), the lingual
antimicrobial peptide (LAP) (26), or hBD-1 (1,
10). The putative mature peptide contains six cysteine residues
spaced in a typical array and other conserved amino acids that may have important roles in determining the conformation and function of -defensins, such as G10, P18, G25, and T26 (33). Several
characteristic charged residues are present in the putative mature
peptide.
Analysis of the mBD-1 gene revealed the presence of two exons
surrounding a 15-kb intron. The structure of the mBD-1 gene is
identical to those of other -defensins, such as LAP (2) or hBD-1 (17), each of which contains two exons. The first
exon contains the prepeptide and part of the propeptide, whereas the mature peptide and the remaining part of the propeptide are encoded by
the second exon. These data indicate an evolutionary conservation of
the members of the -defensin group. Analysis of the cDNA and genomic clones of mBD-1 revealed the presence of typical
regulatory elements, such as a TATA box and a polyadenylation signal;
however, no binding sites for transcription factors that are involved
in the inflammatory response could be found. This differs from the 5'
region of the TAP-encoding gene, which contains an NF-
B site upstream of the transcriptional start site (26). Expression of other mucosal -defensins, such as LAP or TAP, is upregulated by
inflammatory mediators and bacterial components (2, 3, 24,
26). Similar studies of mBD-1 are under way to evaluate the
potential role of regulation in its biology in vivo.
The chromosomal localization of mBD-1 was analyzed by FISH and mapped
to the proximal region of chromosome 8, an area which also contains the
Defcr locus, where genes for mouse -defensins are found
(21). Further, this area is homologous to human chromosome 8p23, where human - and -defensins are located (17).
These results indicate not only a close relationship of mBD-1 and
cryptdins but, further, make evolutionary conservation of the - and
-defensin groups likely. Thus, development of these two groups of
antimicrobial peptides may have taken place during the development of
mammalia before rodents and primates separated.
To further prove that the cloned cDNA sequence encodes an
antimicrobial peptide, the activity of the expressed peptide against different bacteria was analyzed. Lysates of cells transfected with the
mBD-1 cDNA revealed activity against gram-negative and gram-positive
bacteria. Further, this activity was lost at high concentrations of
salt and at an acidic pH. These antimicrobial activities are
characteristic of defensins and are also shared by other known
-defensins (2, 4, 10, 26).
To compare the expression patterns of mBD-1 with those of other known
epithelial -defensins, an RNase protection assay, RT-PCR, and in
situ hybridization were performed. Our data presented here indicate
that mBD-1 is expressed in a pattern similar to that of hBD-1; the site
of the most abundant expression is the distal collecting ducts of the
kidney and other urogenital organs. This suggests that mBD-1 plays a
role in preventing ascending infections such as pyelonephritis in the
kidney and pelvic inflammatory disease in the female reproductive
tract. Expression of mBD-1 is also found throughout the surface
epithelia of the conducting airways. There appears to be a gradient of
mBD-1 expression throughout the conducting airways, with the highest
expression noted in proximal structures. This differs from hBD-1, which
is expressed more uniformly throughout the conducting airways (10,
19). High expression of mBD-1 at sites within the oropharynx
(e.g., the tongue) may also contribute to innate immunity in the lung
by providing proximal defense against inhaled and aspirated organisms.
The expression pattern of mBD-1 in airways is of specific importance in
light of recent findings that implicate a salt-dependent defect of
hBD-1 in the pathogenesis of CF (10, 29). This model
specifically suggests that an elevated concentration of NaCl in the
airway surface fluid of CF patients inactivates the antimicrobial
activity of defensin and/or other antimicrobial molecules, possibly
resulting in bacterial colonization and infection.
In summary, mBD-1 is a murine -defensin diffusely expressed
through the epithelia of multiple organs. It likely plays an important
role in innate immunity at multiple mucosal sites.
| |
ACKNOWLEDGMENTS |
|---|
We thank the Microbiology Laboratory, University of Pennsylvania Health System, for making bacterial cultures available. The contribution of the Cell Morphology Core of the Institute of Human Gene Therapy was greatly appreciated.
This study was supported by the Cystic Fibrosis Foundation, NIDDK, and NHLBI of the NIH, as well as Genovo, Inc. R.B. received a fellowship from The Deutsche Forschungsgemeinschaft.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Molecular and Cellular Engineering, The Institute for Human Gene Therapy, The University of Pennsylvania Medical Center, and The Wistar Institute, Room 204, 3601 Spruce Street, Philadelphia, PA 19104-4268. Phone: (215) 898-3000. Fax: (215) 898-6588. E-mail: jurmu{at}wista.wista.upenn.edu.
Editor: J. R. McGhee
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. |
Bensch, K. W.,
M. Raida,
H.-J. Magert,
P. Schulz-Knappe, and W. G. Forssmann.
1995.
hBD-1: a novel -defensin from human plasma.
FEBS Lett.
368:331-335[Medline].
|
| 2. |
Diamond, G.,
D. E. Jones, and C. L. Bevins.
1993.
Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene.
Proc. Natl. Acad. Sci. USA
90:4596-4600 |
| 3. |
Diamond, G.,
J. P. Russell, and C. L. Bevins.
1996.
Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells.
Proc. Natl. Acad. Sci. USA
93:5156-5160 |
| 4. |
Diamond, G.,
M. Zasloff,
H. Eck,
M. Brasseur,
W. L. Maloy, and C. L. Bevins.
1991.
Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of cDNA.
Proc. Natl. Acad. Sci. USA
88:3952-3956 |
| 5. |
Eisenhauer, P. B.,
S. S. L. Harwig, and R. I. Lehrer.
1992.
Cryptdins: antimicrobial defensins of the murine small intestine.
Infect. Immun.
60:3556-3565 |
| 6. |
Eisenhauer, P. B., and R. I. Lehrer.
1992.
Mouse neutrophils lack defensins.
Infect. Immun.
60:3446-3447 |
| 7. | Evans, E. W., G. G. Beach, J. Wunderlich, and B. G. Harmon. 1994. Isolation of antimicrobial peptides from avian heterophils. J. Leukocyte Biol. 56:661-665[Abstract]. |
| 8. | Ganz, T., and R. I. Lehrer. 1995. Defensins. Pharm. Ther. 66:191-205[Medline]. |
| 9. | Gilljam, H., A. Ellin, and B. Strandvik. 1989. Increased bronchial chloride concentration in cystic fibrosis. Scand. J. Clin. Lab. Invest. 49:121-124[Medline]. |
| 10. |
Goldman, M. J.,
G. M. Anderson,
E. D. Stolzenberg,
U. P. Kari,
M. Zasloff, and J. M. Wilson.
1997.
Human -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.
Cell
88:553-560[Medline].
|
| 11. | Hancock, R. E. W. 1997. Peptide antibiotics. Lancet 349:412-422. |
| 12. | Harwig, S. S. L., K. M. Swiderek, V. N. Kokryakov, L. Tan, T. D. Lee, E. A. Panyutich, E. M. Aleshina, O. V. Shamova, and R. I. Lehrer. 1994. Gallinacins: cystine-rich antimicrobial peptides of chicken leukocytes. FEBS Lett. 342:281-285[Medline]. |
| 13. | Huttner, K. M., M. E. Selsted, and A. J. Ouellette. 1994. Structure and diversity of the murine cryptdin gene family. Genomics 19:448-453[Medline]. |
| 14. |
Jones, D. E., and C. L. Bevins.
1992.
Paneth cells of the human small intestine express an antimicrobial peptide gene.
J. Biol. Chem.
267:23216-23225 |
| 15. | Joris, L., I. Dab, and P. M. Quinton. 1993. Elemental composition of human airway surface fluid in healthy and diseased airways. Am. Rev. Respir. Dis. 148:1633-1637[Medline]. |
| 16. | Lehrer, R. I., M. Rosenman, S. S. Harwig, R. Jackson, and P. Eisenhauer. 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137:167-173[Medline]. |
| 17. |
Liu, L.,
H. Heng,
C. Zhao, and T. Ganz.
1996.
Human - and- -defensins evolved from a common pre-mammalian ancestor peptide.
J. Invest. Med.
44:294A.
|
| 18. |
Mallow, E. B.,
A. Harris,
N. Salzman,
J. P. Russell,
R. J. DeBerardinis,
E. Ruchelli, and C. L. Bevins.
1996.
Human enteric defensins.
J. Biol. Chem.
271:4038-4045 |
| 19. |
McCray, P. B., and L. Bently.
1997.
Human airway epithelia express a -defensin.
Am. J. Respir. Cell Mol. Biol.
16:343-349[Abstract].
|
| 20. |
Mount, S.
1982.
A catalogue of splice junction sequences.
Nucleic Acids Res.
10:459-472 |
| 21. | Ouellette, A. J., D. Pravtcheva, F. H. Ruddle, and M. James. 1989. Localization of the cryptdin locus on mouse chromosome 8. Genomics 5:233-239[Medline]. |
| 22. |
Ouellette, A. J.,
M. M. Hsieh,
M. T. Nosek,
D. F. Cano-Gauci,
K. M. Huttner,
R. N. Buick, and M. E. Selsted.
1994.
Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.
Infect. Immun.
62:5040-5047 |
| 23. | Ouellette, A. J., S. I. Miller, A. H. Henschen, and M. E. Selsted. 1992. Purification and primary structure of murine cryptdin-1, a Paneth cell defensin. FEBS Lett. 304:146-148[Medline]. |
| 24. | Russell, J. P., G. Diamond, A. P. Tarver, T. F. Scanlin, and C. L. Bevins. 1996. Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha. Infect. Immun. 64:1565-1568[Abstract]. |
| 25. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. . Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 26. |
Schonwetter, B. S.,
E. D. Stolzenberg, and M. A. Zasloff.
1995.
Epithelial antibiotics induced at sites of inflammation.
Science
267:1645-1648 |
| 27. |
Selsted, M. E.,
D. Szklarek, and R. I. Lehrer.
1984.
Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes.
Infect. Immun.
45:150-154 |
| 28. |
Selsted, M. E.,
Y. Q. Tang,
W. L. Morris,
P. A. McGuire,
M. J. Novotny,
W. Smith,
A. H. Henschen, and J. S. Cullor.
1993.
Purification, primary structures, and antibacterial activities of -defensins, a new family of antimicrobial peptides from bovine neutrophils.
J. Biol. Chem.
268:6641-6648 |
| 29. | Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. Welsh. 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229-236[Medline]. |
| 30. | Steiner, H., D. Hultmark, A. Engstrom, H. Bennich, and H. G. Boman. 1981. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246-248[Medline]. |
| 31. |
Tang, Y. Q., and M. E. Selsted.
1993.
Characterization of the disulfide motif in BNBD-12, an antimicrobial -defensin peptide from bovine neutrophils.
J. Biol. Chem.
268:6649-6653 |
| 32. | Zhao, C., I. Wang, and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396:319-322[Medline]. |
| 33. |
Zimmerman, G. R.,
P. Legault,
M. E. Selsted, and A. Pardi.
1995.
Solution structure of bovine -defensin-12: the peptide fold of the -defensins is identical to that of the classical defensins.
Biochemistry
34:13663-13671[Medline].
|
Infect Immun, March 1998, p. 1225-1232, Vol. 66, No. 3
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Wilson, C. L., Schmidt, A. P., Pirila, E., Valore, E. V., Ferri, N., Sorsa, T., Ganz, T., Parks, W. C.
(2009). Differential Processing of {alpha}- and {beta}-Defensin Precursors by Matrix Metalloproteinase-7 (MMP-7). J. Biol. Chem.
284: 8301-8311
[Abstract] [Full Text] -
Li, J., Zhu, H. Y., Beuerman, R. W.
(2009). Stimulation of Specific Cytokines in Human Conjunctival Epithelial Cells by Defensins HNP1, HBD2, and HBD3. IOVS
50: 644-653
[Abstract] [Full Text] -
Wu, M., McClellan, S. A., Barrett, R. P., Hazlett, L. D.
(2009). {beta}-Defensin-2 Promotes Resistance against Infection with P. aeruginosa. J. Immunol.
182: 1609-1616
[Abstract] [Full Text] -
Hussain, T., Nasreen, N., Lai, Y., Bellew, B. F., Antony, V. B., Mohammed, K. A.
(2008). Innate immune responses in murine pleural mesothelial cells: Toll-like receptor-2 dependent induction of {beta}-defensin-2 by staphylococcal peptidoglycan. Am. J. Physiol. Lung Cell. Mol. Physiol.
295: L461-L470
[Abstract] [Full Text] -
Taylor, K., McCullough, B., Clarke, D. J., Langley, R. J., Pechenick, T., Hill, A., Campopiano, D. J., Barran, P. E., Dorin, J. R., Govan, J. R. W.
(2007). Covalent Dimer Species of {beta}-Defensin Defr1 Display Potent Antimicrobial Activity against Multidrug-Resistant Bacterial Pathogens. Antimicrob. Agents Chemother.
51: 1719-1724
[Abstract] [Full Text] -
Elahi, S., Buchanan, R. M., Attah-Poku, S., Townsend, H. G. G., Babiuk, L. A., Gerdts, V.
(2006). The Host Defense Peptide Beta-Defensin 1 Confers Protection against Bordetella pertussis in Newborn Piglets. Infect. Immun.
74: 2338-2352
[Abstract] [Full Text] -
Moraes, T. J., Plumb, J., Martin, R., Vachon, E., Cherepanov, V., Koh, A., Loeve, C., Jongstra-Bilen, J., Zurawska, J. H., Kus, J. V., Burrows, L. L., Grinstein, S., Downey, G. P.
(2006). Abnormalities in the Pulmonary Innate Immune System in Cystic Fibrosis. Am. J. Respir. Cell Mol. Bio.
34: 364-374
[Abstract] [Full Text] -
Tydell, C. C., Yuan, J., Tran, P., Selsted, M. E.
(2006). Bovine Peptidoglycan Recognition Protein-S: Antimicrobial Activity, Localization, Secretion, and Binding Properties. J. Immunol.
176: 1154-1162
[Abstract] [Full Text] -
Braff, M. H., Zaiou, M., Fierer, J., Nizet, V., Gallo, R. L.
(2005). Keratinocyte Production of Cathelicidin Provides Direct Activity against Bacterial Skin Pathogens. Infect. Immun.
73: 6771-6781
[Abstract] [Full Text] -
Bowdish, D. M. E., Davidson, D. J., Lau, Y. E., Lee, K., Scott, M. G., Hancock, R. E. W.
(2005). Impact of LL-37 on anti-infective immunity. J. Leukoc. Biol.
77: 451-459
[Abstract] [Full Text] -
Yenugu, S., Richardson, R. T., Sivashanmugam, P., Wang, Z., O'Rand, M. G., French, F. S., Hall, S. H.
(2004). Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif. Biol. Reprod.
71: 1484-1490
[Abstract] [Full Text] -
Yenugu, S., Hamil, K. G., Radhakrishnan, Y., French, F. S., Hall, S. H.
(2004). The Androgen-Regulated Epididymal Sperm-Binding Protein, Human {beta}-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial {beta}-Defensin. Endocrinology
145: 3165-3173
[Abstract] [Full Text] -
Zaalouk, T. K., Bajaj-Elliott, M., George, J. T., McDonald, V.
(2004). Differential Regulation of {beta}-Defensin Gene Expression during Cryptosporidium parvum Infection. Infect. Immun.
72: 2772-2779
[Abstract] [Full Text] -
Yamamoto, C. M., Banaiee, N., Yount, N. Y., Patel, B., Selsted, M. E.
(2004). {alpha}-Defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes. J. Leukoc. Biol.
75: 332-341
[Abstract] [Full Text] -
Cunliffe, R. N., Mahida, Y. R.
(2004). Expression and regulation of antimicrobial peptides in the gastrointestinal tract. J. Leukoc. Biol.
75: 49-58
[Abstract] [Full Text] -
Morrison, G. M., Semple, C. A. M., Kilanowski, F. M., Hill, R. E., Dorin, J. R.
(2003). Signal Sequence Conservation and Mature Peptide Divergence Within Subgroups of the Murine {beta}-Defensin Gene Family. Mol Biol Evol
20: 460-470
[Abstract] [Full Text] -
Yamaguchi, Y., Nagase, T., Makita, R., Fukuhara, S., Tomita, T., Tominaga, T., Kurihara, H., Ouchi, Y.
(2002). Identification of Multiple Novel Epididymis-Specific {beta}-Defensin Isoforms in Humans and Mice. J. Immunol.
169: 2516-2523
[Abstract] [Full Text] -
Morrison, G., Kilanowski, F., Davidson, D., Dorin, J.
(2002). Characterization of the Mouse Beta Defensin 1, Defb1, Mutant Mouse Model. Infect. Immun.
70: 3053-3060
[Abstract] [Full Text] -
Moser, C., Weiner, D. J., Lysenko, E., Bals, R., Weiser, J. N., Wilson, J. M.
(2002). {beta}-Defensin 1 Contributes to Pulmonary Innate Immunity in Mice. Infect. Immun.
70: 3068-3072
[Abstract] [Full Text] -
SCHALLER-BALS, S., SCHULZE, A., BALS, R.
(2002). Increased Levels of Antimicrobial Peptides in Tracheal Aspirates of Newborn Infants during Infection. Am. J. Respir. Crit. Care Med.
165: 992-995
[Abstract] [Full Text] -
Ayabe, T., Wulff, H., Darmoul, D., Cahalan, M. D., Chandy, K. G., Ouellette, A. J.
(2002). Modulation of Mouse Paneth Cell alpha -Defensin Secretion by mIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel. J. Biol. Chem.
277: 3793-3800
[Abstract] [Full Text] -
Schroeder, T. H., Reiniger, N., Meluleni, G., Grout, M., Coleman, F. T., Pier, G. B.
(2001). Transgenic Cystic Fibrosis Mice Exhibit Reduced Early Clearance of Pseudomonas aeruginosa from the Respiratory Tract. J. Immunol.
166: 7410-7418
[Abstract] [Full Text] -
Zhao, C., Nguyen, T., Liu, L., Sacco, R. E., Brogden, K. A., Lehrer, R. I.
(2001). Gallinacin-3, an Inducible Epithelial {beta}-Defensin in the Chicken. Infect. Immun.
69: 2684-2691
[Abstract] [Full Text] -
Li, P., Chan, H. C., He, B., So, S. C., Chung, Y. W., Shang, Q., Zhang, Y.-D., Zhang, Y.-L.
(2001). An Antimicrobial Peptide Gene Found in the Male Reproductive System of Rats. Science
291: 1783-1785
[Abstract] [Full Text] -
Bals, R., Lang, C., Weiner, D. J., Vogelmeier, C., Welsch, U., Wilson, J. M.
(2001). Rhesus Monkey (Macaca mulatta) Mucosal Antimicrobial Peptides Are Close Homologues of Human Molecules. CVI
8: 370-375
[Abstract] [Full Text] -
Paulsen, F P, Tschernig, T, Debertin, A S, Kleemann, W J, Pabst, R, Tillmann, B N
(2001). Similarities and differences in lectin cytochemistry of laryngeal and tracheal epithelium and subepithelial seromucous glands in cases of sudden infant death and controls. Thorax
56: 223-227
[Abstract] [Full Text] -
Kaiser, V., Diamond, G.
(2000). Expression of mammalian defensin genes. J. Leukoc. Biol.
68: 779-784
[Abstract] [Full Text] -
Haversen, L. A., Engberg, I., Baltzer, L., Dolphin, G., Hanson, L. A., Mattsby-Baltzer, I.
(2000). Human Lactoferrin and Peptides Derived from a Surface-Exposed Helical Region Reduce Experimental Escherichia coli Urinary Tract Infection in Mice. Infect. Immun.
68: 5816-5823
[Abstract] [Full Text] -
Lopez-Boado, Y. S., Wilson, C. L., Hooper, L. V., Gordon, J. I., Hultgren, S. J., Parks, W. C.
(2000). Bacterial Exposure Induces and Activates Matrilysin in Mucosal Epithelial Cells. JCB
148: 1305-1315
[Abstract] [Full Text] -
Yu, Q., Lehrer, R. I., Tam, J. P.
(2000). Engineered Salt-insensitive alpha -Defensins with End-to-end Circularized Structures. J. Biol. Chem.
275: 3943-3949
[Abstract] [Full Text] -
BEVINS, C L, MARTIN-PORTER, E, GANZ, T
(1999). Defensins and innate host defence of the gastrointestinal tract. Gut
45: 911-915
[Full Text] -
Yount, N. Y., Yuan, J., Tarver, A., Castro, T., Diamond, G., Tran, P. A., Levy, J. N., McCullough, C., Cullor, J. S., Bevins, C. L., Selsted, M. E.
(1999). Cloning and Expression of Bovine Neutrophil beta -Defensins. BIOSYNTHETIC PROFILE DURING NEUTROPHILIC MATURATION AND LOCALIZATION OF MATURE PEPTIDE TO NOVEL CYTOPLASMIC DENSE GRANULES. J. Biol. Chem.
274: 26249-26258
[Abstract] [Full Text] -
Jia, H. P., Mills, J. N., Barahmand-Pour, F., Nishimura, D., Mallampali, R. K., Wang, G., Wiles, K., Tack, B. F., Bevins, C. L., McCray, P. B. Jr.
(1999). Molecular Cloning and Characterization of Rat Genes Encoding Homologues of Human beta -Defensins. Infect. Immun.
67: 4827-4833
[Abstract] [Full Text] -
Zhang, G., Hiraiwa, H., Yasue, H., Wu, H., Ross, C. R., Troyer, D., Blecha, F.
(1999). Cloning and Characterization of the Gene for a New Epithelial beta -Defensin. GENOMIC STRUCTURE, CHROMOSOMAL LOCALIZATION, AND EVIDENCE FOR ITS CONSTITUTIVE EXPRESSION. J. Biol. Chem.
274: 24031-24037
[Abstract] [Full Text] -
Bals, R., Wang, X., Meegalla, R. L., Wattler, S., Weiner, D. J., Nehls, M. C., Wilson, J. M.
(1999). Mouse beta -Defensin 3 Is an Inducible Antimicrobial Peptide Expressed in the Epithelia of Multiple Organs. Infect. Immun.
67: 3542-3547
[Abstract] [Full Text] -
McCray, P. B. Jr., Zabner, J., Jia, H. P., Welsh, M. J., Thorne, P. S.
(1999). Efficient killing of inhaled bacteria in Delta F508 mice: role of airway surface liquid composition. Am. J. Physiol. Lung Cell. Mol. Physiol.
277: L183-L190
[Abstract] [Full Text] -
Shi, J., Zhang, G., Wu, H., Ross, C., Blecha, F., Ganz, T.
(1999). Porcine Epithelial beta -Defensin 1 Is Expressed in the Dorsal Tongue at Antimicrobial Concentrations. Infect. Immun.
67: 3121-3127
[Abstract] [Full Text] -
Ouellette, A. J., Satchell, D. P., Hsieh, M. M., Hagen, S. J., Selsted, M. E.
(2000). Characterization of Luminal Paneth Cell alpha -Defensins in Mouse Small Intestine. ATTENUATED ANTIMICROBIAL ACTIVITIES OF PEPTIDES WITH TRUNCATED AMINO TERMINI. J. Biol. Chem.
275: 33969-33973
[Abstract] [Full Text] -
Jia, H. P., Wowk, S. A., Schutte, B. C., Lee, S. K., Vivado, A., Tack, B. F., Bevins, C. L., McCray, P. B. Jr.
(2000). A Novel Murine beta -Defensin Expressed in Tongue, Esophagus, and Trachea. J. Biol. Chem.
275: 33314-33320
[Abstract] [Full Text] -
Yamaguchi, Y., Fukuhara, S., Nagase, T., Tomita, T., Hitomi, S., Kimura, S., Kurihara, H., Ouchi, Y.
(2001). A Novel Mouse beta -Defensin, mBD-6, Predominantly Expressed in Skeletal Muscle. J. Biol. Chem.
276: 31510-31514
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»