U.S. patent application number 09/991792 was filed with the patent office on 2002-07-25 for antisecretory factor peptides regulating pathological permeability changes.
Invention is credited to Jennische, Eva, Johansson, Eva, Lange, Stefan, Lonnroth, Christina, Lonnroth, Ivar.
Application Number | 20020099016 09/991792 |
Document ID | / |
Family ID | 20399269 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020099016 |
Kind Code |
A1 |
Lonnroth, Ivar ; et
al. |
July 25, 2002 |
Antisecretory factor peptides regulating pathological permeability
changes
Abstract
A new recombinant protein called Antisecretory Factor (rAF) and
homologues and peptide fragments thereof are described. The protein
and the homologues and fragments thereof are useful for normalising
pathological fluid transport and/or inflammatory reactions in
animals including humans. Antibodies against AF or homologues or
fragments thereof are described. Nucleic acids coding for the
protein or for homologues or fragments thereof are also described
as well as vectors and hosts comprising the nucleic acids. The rAF
and homologues and fragments thereof could be used for
immunodetection, as feed additive for growing animals and as
antidiarrheal and drugs against diseases involving edema,
dehydration and/or inflammation.
Inventors: |
Lonnroth, Ivar; (Molndal,
SE) ; Lange, Stefan; (Goteborg, SE) ;
Johansson, Eva; (Molndal, SE) ; Jennische, Eva;
(Goteborg, SE) ; Lonnroth, Christina; (Molndal,
SE) |
Correspondence
Address: |
Benton S. Duffett, Jr.
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
20399269 |
Appl. No.: |
09/991792 |
Filed: |
November 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09991792 |
Nov 26, 2001 |
|
|
|
09029333 |
Mar 13, 1998 |
|
|
|
09029333 |
Mar 13, 1998 |
|
|
|
PCT/SE96/01049 |
Aug 23, 1996 |
|
|
|
Current U.S.
Class: |
514/5.5 ;
514/12.2; 530/324 |
Current CPC
Class: |
A61P 1/12 20180101; A61P
43/00 20180101; C07K 14/575 20130101; A61P 17/00 20180101; Y02A
50/30 20180101; A61P 3/00 20180101; A23K 20/147 20160501; A61P
25/36 20180101; A61P 13/12 20180101; A61K 38/00 20130101; A61P
27/02 20180101; A61P 3/10 20180101; A61P 11/06 20180101; A61P 25/02
20180101; A61P 5/40 20180101; A61P 1/08 20180101; A61P 25/24
20180101; A61P 1/04 20180101; A61P 27/06 20180101; A61P 1/00
20180101; C07K 14/47 20130101; A61P 19/02 20180101; A61P 5/00
20180101; A61P 27/16 20180101; A61P 29/00 20180101; A61P 7/12
20180101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 038/16; C07K
014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 1995 |
SE |
9502936-9 |
Claims
We claim:
1. A synthetic protein comprising SEQ ID NO:2 or a homolog or a
fragment thereof, and wherein said homolog or fragment of SEQ ID
NO:2 comprises formula X.sub.1VCX.sub.2X.sub.3KX.sub.4R, (a)
wherein said formula corresponds to amino acids 35-42 of SEQ ID
NO:2; (b) X.sub.1 is I or is absent; (c) X.sub.2 is H, R or K; (d)
X.sub.3 is S, L or anther neutral amino acid; and (d) X.sub.4 is T
or A; and wherein said synthetic protein or homolog or fragment
thereof has antisecretory activity when administered after cholera
toxin challenge.
2. A composition comprising the synthetic protein or homolog or
fragment of claim 1.
3. The synthetic protein of claim 1, wherein said synthetic protein
consists of SEQ ID NO:2.
4. A composition comprising the synthetic protein of claim 3.
5. A synthetic polypeptide comprising (a) amino acids 35-42, (b)
amino acids 35-46, (c) amino acids 36-51, (d) amino acids 36-80, or
(e) amino acids 1-80 of SEQ ID NO:2, or (f) a peptide of formula
X.sub.1VCX.sub.2X.sub.3KX.sub.4R wherein the formula corresponds to
amino acids 35-42 of SEQ ID NO:2 of any of polypeptides (a) to (e),
and wherein (i) X.sub.1 is I or is absent; (ii) X.sub.2 is H, R or
K; (iii) X.sub.3 is S, L or anther neutral amino acid; and (iv)
X.sub.4 is T or A.
6. A composition comprising the synthetic polypeptide of claim
5.
7. A synthetic polypeptide consisting of a sequence of amino acids
selected from the group consisting of (a) amino acids 35-42, (b)
amino acids 35-46, (c) amino acids 36-51, (d) amino acids 36-80,
and (e) amino acids 1-80 of SEQ ID NO:2.
8. A composition for use in vertebrates including humans comprising
an effective amount of the synthetic protein or fragment or homolog
of claim 1, wherein said composition has antisecretory
activity.
9. A synthetic composition for use in vertebrates including humans
comprising an effective amount of the synthetic polypeptide of
claim 5, wherein said composition has antisecretory activity.
10. A method of using the synthetic protein or homolog or fragment
of claim 1 comprising administering an effective amount of the
synthetic protein or homolog or fragment to a vertebrate to induce
antisecretory activity.
11. A method of using the synthetic polypeptide of claim 5
comprising administering an effective amount of the synthetic
polypeptide to a vertebrate to induce antisecretory activity.
12. A method of inhibiting diarrhea in a vertebrate comprising
administering the composition of claim 2.
13. A method of inhibiting diarrhea in a vertebrate comprising
administering the composition of claim 6.
14. The method of claim 12, wherein said vertebrate is a human.
15. The method of claim 13, wherein said vertebrate is a human.
16. A feed or food for vertebrates including humans comprising an
active agent, wherein the active agent is the synthetic protein or
a homolog or a fragment thereof of claim 1 wherein said feed or
food has antisecretory activity.
17. A feed or food for vertebrates comprising an active agent,
wherein said active agent is the synthetic polypeptide of claim 5
and wherein said feed or food has antisecretory activity.
18. A feed additive comprising the synthetic protein or homolog or
fragment thereof of claim 1, wherein said feed additive has
antisecretory activity.
19. A feed additive comprising the synthetic polypeptide of claim
5, wherein said feed additive has antisecretory activity.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/029,333, filed on Mar. 13, 1998, which was a national stage
filing under 35 U.S.C. .sctn. 371 of International Application No.
PCT/SE96/01049 filed on Aug. 23, 1996, which International
Application was published by the International Bureau in English on
Mar. 6, 1997, and which claims priority to Swedish Application No.
9502936-9 filed Aug. 24, 1995.
[0002] The present invention relates to new antisecretory factors
having fluid transport and/or inflammatory reactions regulating
properties as well as polynucleic regulating properties, and
polynucleic acids coding therefor, and the use thereof.
[0003] All cells and tissues of the body are critically dependent
on a constant and normal fluid environment in combination with an
adequate blood supply. Derangement of one or both of these
supporting systems may rapidly become fatal. Concerning fluid
imbalance, two principally different systems exist:
[0004] A. edema, which is characterized by the abnormal
accumulation of fluid in the intercellular tissue spaces or body
cavities, or
[0005] B. dehydration, which, in a strict sense, means loss of
water only, but is in fact commonly used to describe the combined
loss of water and ions.
[0006] The most common forms of either edema or dehydration are:
diarrheas, inflammatory bowel diseases, brain edema, asthma,
rhinitis, conjunctivitis, arthritis, glaucoma, various forms of
pathological intracranial pressure (increase or decrease), pressure
alteration in the middle ear such as Morbus Mnire, dermatitis,
chemical or physical derangement of the skin and skin adjacent
glands such as mastitis, various forms of endocrine disorders, such
as diabetes insipidus. Conn's syndrome, Cushing's syndrome and
Morbus Addison, kidney diseases such as pyslonephritis and
glomerulonephritis, metabolic diseases such as myxedema and acute
intermittent porphyria, side effects during treatment with various
drugs such as antidiabetics, tricyclic antidepressants,
cytostatics, barbiturates, narcotics and narcotic analogues.
[0007] Diarrhea is caused by a change in the permeability in the
gut for electrolytes and water. This disturbance is often caused by
bacterial enterotoxins such as those produced by Escherichia coli,
Campylobacter jejuni, Vibrio cholerae, Shigella dysenteriae and
Chostridium difficile. The disturbance could also be caused by
intestinal inflammation. Since the uptake of water is coupled to
the uptake of electrolytes and nutrients, animals with frequent
diarrhea suffers from malnutrition, resulting in retardation of the
daily weight gain in the growing animal. The body counteracts these
reactions by neuro-hormonal mechanisms such as the release of
somatostatin and opiate peptides from interneurons in the
intestinal mucosa. These polypeptides are capable of reversing
fluid secretion and diarrhea.
[0008] The recently described antisecretory factor (AF) has been
partially purified from pig pituitary gland and shown to reverse
pathological secretion induced by various enterotoxins. High levels
of AF in sow milk protect the suckling piglets against neonatal
diarrhea.
[0009] Antimicrobial drugs have been widely used in the treatment
of diarrhea in both human and veterinarian medicine. They are also
used as feed additives for pigs, calves and chicken. However, due
to the rapid development of resistant bacteria in the gut, the use
of antibiotics against enteritis is generally not accepted in human
medicine and their use is also diminishing in veterinarian
medicine.
[0010] Other antidiarrheal drugs counteract the secretion in the
intestinal mucosa. Since these drugs are directed against the host
animal, it is unlikely that resistance against the drugs will
develop. These types of drugs include nerve-active drugs like
phenothiazines and thioxanthenes. Due to some serious side effects
these types of drugs have not been accepted for treatment of
diarrhea in most countries. Other drugs are derivatives of opiates
like codeine and loperamide since these drugs mainly acts by
inhibiting intestinal mobility, they also inhibit the clearance of
pathogenic bacteria from the gut and should definitely not be
recommended against dysenteric bacteria or parasites. Derivatives
of somatostatin have been introduced recently, but have so far a
limited use due to difficulties in the administration of the drugs
and possible interactions with the endocrine regulation of
growth.
[0011] The antisecretory factor (AF) has so far not been used
directly for treatment of diarrhea or malnutrition due to the
difficulties involved in obtaining a pure preparation of this
protein. However, it has been possible to induce similar proteins
in domestic animals which have been given a specific feed (SE
Patent No. 9000028-2). Pigs given this feed obtained high levels of
AF-like proteins and had a significant increase in the daily growth
rate compared to matched controls. AF in rats challenged with toxin
A from C. difficile protects not only against intestinal secretion
but also against inflammation and bleeding in the gut.
[0012] A major object of the present invention is to provide a new
recombinant protein and homologues and fragments (peptides) thereof
for use in normalizing pathological fluid transport. These proteins
and peptides are collectively called antisecretory factors (AF).
The use of AF also partly inhibits, or totally eliminates the
development of inflammatory reactions of various aetiologies.
Reconstitution back to normal (fluid transport or inflammation) is
obtained by the use of proteins or peptides. Further the AF
proteins or peptides are effectively absorbed via various mucus
membranes without losing in potency (when compared to intravenous
administration). Consequently, a multitude of treatment regimens
exist, and a correctly administrated protein or peptide make it
possible to rapidly reconstitute a deranged fluid (water and ion)
balance, an inflammatory reaction, or both.
[0013] In summary, the recombinant AF (rAF) and the homologues and
fragments thereof could be used for immunodetection, as feed
additive for growing animals and as antidiarrheal and drugs against
diseases involving edema, dehydration and/or inflammation.
[0014] The objects of the present invention are the following:
[0015] A recombinant protein having essentially the amino acid
sequence shown in SEQ ID NO:2, or homologues or fragments
thereof.
[0016] A fragment of the recombinant protein shown in SEQ ID NO:2:
which fragment is chosen from the group comprising
[0017] a) amino acids nos. 35-42
[0018] b) amino acids nos. 35-46
[0019] c) amino acids nos. 36-51
[0020] d) amino acids nos. 36-80
[0021] e) amino acids nos. 1-80
[0022] of the amino acid sequence shown in SEQ ID NO:2.
[0023] A peptide X.sub.1VCX.sub.2X.sub.3KX.sub.4R corresponding to
the fragment comprising the amino acids no. 35-42 of the
recombinant protein shown in SEQ ID NO:2, wherein X is I or none,
X.sub.2 is H, R or K, X.sub.3 is S, L or another neutral amino acid
and X.sub.4 is T or A.
[0024] Antibodies against a recombinant protein having essentially
the amino acid sequence shown in SEQ ID NO:2, or homologues or
fragments thereof.
[0025] A protein binding to antibodies specific to a recombinant
protein having essentially the amino acid sequence shown in SEQ ID
No. 1, or homologues or fragments thereof.
[0026] A composition for normalizing pathological fluid transport
and/or inflammatory reactions comprising as an active principal an
effective amount of the recombinant protein having essentially the
amino acid sequence shown in SEQ ID NO:2, or homologues or
fragments thereof.
[0027] Use of a recombinant protein having essentially the amino
acid sequence shown in SEQ ID NO:2, homologues or fragments thereof
for manufacturing a composition for normalizing pathological fluid
transport and/or inflammatory reactions.
[0028] Feed for normalizing pathological fluid transport and/or
inflammatory reactions in vertebrates, comprising as an active
agent a recombinant protein having essentially the amino acid
sequence shown in SEQ ID NO:2, or homologues or fragments thereof,
or an organism capable of producing such a protein or homologues or
fragments thereof.
[0029] A process of normalizing pathological fluid transport and/or
inflammatory reactions in vertebrates, comprising administering to
the vertebrate an effective amount of a recombinant protein having
essentially the amino acid sequence shown in SEQ ID NO: 1, or
homologues or fragments thereof, or an organism producing said
protein or homologues or fragments.
[0030] Use of specific antibodies against a recombinant protein
having essentially the amino acid sequence shown in SEQ ID NO:2, or
homologues or fragments thereof, for detecting said protein or
fragments in organisms.
[0031] Nucleic acids coding for a recombinant protein having
essentially the sequence shown in SEQ ID NO:2, or homologues or
fragments thereof.
[0032] Use of nucleic acids coding for a recombinant protein having
essentially the amino acid sequence shown in SEQ ID No. 1, or
homologues or fragments thereof, for producing corresponding
proteins or homologues or fragments.
[0033] Use of probes or primers derived from nucleic acids coding
for a recombinant protein having essentially the sequence shown in
SEQ. ID No. 1, or homologues or fragments thereof, for detecting
the presence of nucleic acids in organisms.
[0034] Vector comprising nucleic acids coding for a recombinant
protein having essentially the amino acid sequence shown in SEQ. ID
No. 1, or homologues or fragments thereof.
[0035] Host except human comprising a vector including nucleic
acids coding for a recombinant protein having essentially the amino
acid sequence shown in SEQ. ID No. 1, or homologues or fragments
thereof.
[0036] A strain of an organism except human capable of producing a
protein having essentially the amino acid sequence shown in SEQ ID
No. 1, or homologues or fragments thereof.
[0037] As organisms capable of producing the recombinant protein
use can be made of different types of organisms, such as
recombinant bacteria and eucaryotic organisms, such as yeast,
plants and vertebrates except humans.
[0038] Despite ten years of attempts to purify AF by conventional
biochemical techniques, it has not been possible to obtain AF in a
homogeneous form. However, by means of a new procedure of preparing
a semipure AF for immunization and selecting antiserum by means of
an immunohistochemical method a suitable antiserum was chosen. With
this antiserum it has now been possible to clone recombinant human
cDNA expressing AF in E. coli.
[0039] The sequence of the new cDNA was determined and shown to be
unique. By knowledge of this sequence, oligonucleotide probes were
constructed which hybridize with human and porcine pituitary RNA.
The size of this RNA, about 1400 basepairs, complies with the size
of the sequenced cDNA comprising 1309 basepairs plus a poly(A)tail.
A partial cDNA sequence from rat pituitary gland has been shown to
be identical with that of the human cDNA reflecting a ubiquitous
structure conserved in AF genes from different species. This
resemblance makes it possible to use the same oligonucleotide
probes to identify AF-coding RNA and DNA from different
species.
[0040] It has furthermore been possible to express the rAF in a
biological active form. The AF protein in form of a fusion protein
with gluthathione S-transferase was expressed in large amounts in
E. coli. and purified to homogeneity by affinity chromatography.
After cleavage of the fusion protein with thrombin, the recombinant
AF (rAF) was shown to be extremely potent, 44 ng (10.sup.-12 mol)
giving a half-maximal inhibition of cholera toxin-induced fluid
secretion in rat intestine.
[0041] By gene technique smaller fragments of rAF was produced. The
activity was shown to reside in a small sequence consisting of 7 to
8 amino acids. This was confirmed by help of chemical solid phase
synthesis by which technique an octapeptide was produced and shown
to be almost as biological potent as rAF on molar basis. With help
of site directed synthesis a variety of sequences within the active
site was constructed and replacements of certain amino acids shown
to be possible without abolishing the biological activity.
[0042] The fluid secretion was measured by the intestinal loop
model: a section (loop) of the small intestine is ligated by means
of two sutures; in the loop a certain amount of enterotoxin is
injected. If antisecretory drugs are tested they are injected
between one hour before and two hours after toxin challenge. The
injection was made by three different routes; intravenously,
intraintestinally and intranasally. The fluid is accumulating in
the loop 5 h after toxin challenge. The secretion is calculated
from the weight of the accumulated fluid per cm intestine.
[0043] The sequence of the protein was determined both directly by
amino acid sequencing and indirectly by deduction from the cDNA
sequence.
[0044] Recombinant AF seems to exert very little toxic or systemic
effects since no obvious toxic reactions were noted in rats given
100 fold higher doses than that causing half-maximal inhibition.
Since it is efficient when injected in the small intestine it could
be administrated perorally.
[0045] The recombinant AF inhibits secretion also when injected
after toxin challenge in contrast to the preparations of natural AF
tested which seem to efficient only when injected before the toxin.
Thus, rAF could be used both prophylactically and
therapeutically.
[0046] Further, rAF and its peptide fragments were shown to inhibit
cytotoxic reactions and inflammation in the gut caused by toxin A
from Clostridium difficile. By help of a dye permeability test rAF
and its fragments were shown to reverse pathological permeability
changes induced by cholera toxin not only in the intestinal mucosa
but also in plexus choroideus which regulates the fluid pressure in
the brain.
[0047] Antisera against rAF were produced in rabbits and used in
enzyme-linked immuno assays (ELISA). This assay might be used to
measure AF in body fluids or feed.
[0048] A method of purifying antibodies against AF (natural or
recombinant) by means of affinity chromatography on columns with
agarose coupled rAF is reported below.
[0049] The antibodies were also shown to be efficient for detection
of AF in tissue sections by means of immunohistochemical techniques
and for detection of AF in Western-blot.
[0050] The invention will now be described further by means of the
following non-limiting Examples together with the accompanying
drawings.
EXAMPLE 1
Antibodies Against AF Produced for Cloning of cDNA
[0051] Antisecretory factor was prepared from pig blood by means of
affinity chromatography on agarose and isoelectric focusing. To one
liter of pig blood (containing anticoagulating substances) 1 g of
sodium thiosulfate and 1 mg of phenylmethylsulfonylfluoride were
added. The blood cells were separated by centrifugation and the
clear plasma was eluted through a column with Sepharose 6B
(Pharmacia LKB Biotechnology Stockholm), the gel volume
corresponding to about 10% of the volume of the solution. After
washing with three bed volumes of phosphate buffered saline
(PBS=0.15 M NaCl, 0.05 M sodium phosphate, pH 7.2), the column was
eluted with two bed volumes of 1 M .alpha.-methyl-D-glucoside
dissolved in PBS. The eluate was concentrated and dialyzed against
water on an "Omega 10k flow through" ultrafilter (Filtran
Technology Corp.). The fraction was subsequently fractionated by
isoelectric focusing in an ampholine (Pharmacia) gradient pH 4-6 on
a 400 ml isoelectrofocusing column (LKB, Sweden). A fraction having
an isoelectric point-between 4.7 and 4.9 was collected and dialyzed
against PBS. Thus, partially purified AF was divided into small
aliquotes and used for production of antiserum in rabbits according
to a previously described method.
[0052] The rabbits were immunized and the sera tested for their
capacity to stain intracellular material in sections of human
pituitary gland (method described in Example 6). Only one of the
sera showed specific and distinct intracellular staining without
staining extracellular matrix proteins. This antiserum was selected
for screening of a cDNA/lambda phage GT11 library from human
pituitary gland expressing proteins in E. coli.
EXAMPLE 2
Screening cDNA Libraries from Human Pituitary Gland and Brain
[0053] A 5'-stretch cDNA library from normal human pituitary gland,
derived from tissues obtained from a pool of nine Caucasians, was
purchased from Clontech Laboratories. For screening of the library,
phages were plated at 3.times.10.sup.4 plaque forming units per 150
mm dish on E. coli Y1090. The previously described rabbit antiserum
against porcine AF was absorbed with 0.5 volumes of E. coli
Y1090-lysate for 4 hours at 23.degree. C. and diluted to a ratio of
1:400 and screening performed according to Young and Davis (1).
[0054] Alkaline-phosphatase-conjugated goat anti-rabbit antibodies
were used as second antibodies (Jackson). Positive plaques were
picked, eluted into phage suspension medium [20 mM Tris-HCl (pH
7.5), 100 mM NaCl, 10 mM MgSO.sub.4, 2% gelatin], replated, and
screened until all plagues tested were positive.
[0055] cDNA-recloning
[0056] Phage DNA from AF recombinants was isolated with Wizard
Lambda Preps (Promega) and digested with EcoR1. The inserts were
purified with Sephaglas BandPrep Kits (Pharmacia), recloned into
pGex-1.lambda.T vector (Pharmacia) as described by the manufacturer
and transfected into Epicurian Coli XLI-Blue, Top 1 cells or BL21
cells (all three from Stratagen). rAF or rpeptides were prepared in
BL21 cells when not stated otherwise (2).
[0057] Amplification of cDNA by PCR
[0058] To obtain the missing 5'-end of the cDNA a PCR-based method
called RACE (rapid amplification of cDNA ends) was performed. A
modified RACE-method that generates 5'-RACE-Ready cDNA with an
anchor oligonucleotide ligated to the 3'-ends of the human brain
cDNA molecules was purchased from Clontech Laboratories. The 5'-end
was amplified from a portion of the 5'-RACE-Ready cDNA in two PCR
amplification steps using a 5' primer complementary to the anchor
and two nested gene-specific 3 PCR primers A and B (A=base 429-411
and B=base 376-359; FIG. 1a). Various smaller portions of the RACE
fragment was further amplified in order to express the
corresponding peptides and test for their biological properties.
The position of the base and amino acid at the start and end of
these oligonucleotide fragments and their corresponding peptides
are shown in Table 1. Porcine and bovine cDNA (Clontech
Laboratories) was used as templet for amplifying fragments
corresponding to N3 in Table 1. Variation of the sequence was also
inserted artificially by site directed mutagenesis in which method
various oligonucleotides corresponding to position 168-193 was
synthesized in order to replace one by one of amino acid 35-42
(positions as shown in SEQ ID NO:2). The amplified DNA fragment was
cloned into pGex-1.lambda.T vector by using the EcoR1 site built
into the anchor and the gene-specific primer. To verily the
sequence obtained by the RACE method, double stranded cDNA from
human pituitary gland and brain (Clontach) were amplified with
primer pair C/D containing an extra EcoR1-cleavage site (Fig. 1b).
The primers were designed to allow the entire open reading frame
(ORF) to be amplified. The pituitary and brain PCR-products of
expected size were digested with ECOR1, isolated and cloned into
the plasmid pGex-1.lambda.T vector.
[0059] DNA sequencing and oligonucleotides
[0060] DNA from plasmid pGex-1.lambda.T was used as a template for
sequencing of the inserts by dideoxy-chain-termination method (15)
using the Sequence version 2.0 kit (U.S. Biochemical Corp.).
Initial forward and reverse primers copying regions of
pGex-1.lambda.T immediately upstream and downstream of inserted DNA
were obtained from Pharmacia. Subsequent primers were synthesized
(Scandinavian Gene Synthesis AB) on the basis of sequence
information obtained. Three different PCR clones were sequenced in
order to avoid base-exchange by Taq polymerase in the 5'-RACE
method.
[0061] Nucleotide sequence and the deduced protein sequence data
were compiled and analyzed by using MacVector 4.1 (Eastman Chemical
Co.). To predict the corresponding amino acid sequence of the cDNA
inserts, codon usage of different reading frames was compared and
gave one large open reading frame. Interrogation of DNA and protein
sequence data was carried out by use of an Entrez CD-ROM disc
(National Center for Biotechnology Information, Bethesda, USA).
[0062] Molecular cloning and sequence analysis of cDNA
[0063] Polyvalent antisera against AF protein from pig were used
for screening cDNA from human pituitary glands. Two clones
expressing immunoreactive AF were isolated, rescued from phage
lambda and recloned into the EcoR1 site of vector pGex-1.lambda.T
as described in the kit provided from Pharmacia. Restriction
analysis gave insert sizes of 1100 and 900 bp, respectively
DNA-sequencing of the two clones revealed homology to be complete
except for one substitution (FIG. 1, C replacing T at position
1011). A sequence upstream of the 5'-end of clone 2 was obtained by
means of the RACE method. The fragment had a total length of 376 bp
(not including the synthetic nucleotide arm at the 5'-end). The
total reconstructed CDNA contained 1309 basepairs followed a poly-A
tail, which was preceded by a poly-A signal (FIG. 1, positions
1289-1295). An open reading frame (ORF) of 1146 bp (positions
63-1208) was identified.
EXAMPLE 3
Expression of Mammalian AF Protein from Recombinant Plasmids
[0064] Construction and Purification of Fusion Proteins
[0065] The cDNA-clones obtained by immunological screening and by
PCR amplification of the entire CDNA were ligated to
pGex-1.lambda.T. This vector allows expression of foreign proteins
in E. coli as fusions to the C terminus of the Schistosoma
japonicum 26 kDa glutathione S-transferase (GST), which can be
affinity purified under nondenaturing conditions with help of the
kit provided from Pharmacia. Briefly, overnight cultures of E. coli
transformed with recombinant pGex-1.lambda.T plasmids were diluted
in fresh medium and grown for a further 3 h at 37.degree. C.
Protein expression was induced by 0.1 mM IPTG
(isopropyl-beta-D-thiogalac- topyranoside), and after a further 4 h
of growth at 30.degree. C., the calls were pelleted and resuspended
in PBS. Cells were lyzed by sonication, treated with 1% Triton
X-100 and centrifuged at 12000X g for 10 min; the supernatant
containing the expressed fusion proteins was purified by passing
the lysates through glutathione agarose (Pharmacia). The fusion
proteins were either eluted by competition with free glutathione or
were cleaved overnight with 10 U bovine thrombin to remove the
AF-protein from the GST affinity tail. The entire method of using
the pGex plasmid and purifying the recombinant proteins or peptides
was a performed by means of the kits provided from Pharmacia.
[0066] Sequence and size of Recombinant AF-proteins
[0067] To confirm the coding sequence, the full-length transcript
was isolated by using PCR-amplification of pituitary and brain
cDNA. Using the primer pair C/D, 1215 bp identical to the sequence
of clone-4 (FIG. 1, SEQ ID NO: 1) was isolated. The open-reading
frame encoded 382 amino acids with a calculated molecular mass of
41.14 kDa and a calculated pI of 4.9.
[0068] The AF clones-1, 2 and 3 as well as the oligonucleotides
N1-N5 (FIG. 1 and Table 1) were ligated into the pGEX-1.lambda.T
plasmid vector so that the ORF was in frame with the glutathione
S-transferase (GST) protein. The constructs were transformed into
E. coli, and expression of fusion proteins was induced with IPTG.
The purified fusion proteins and the thrombin-cleaved AF protein or
peptide were subjected to SDS-PACE and Western blotting using
antiserum against porcine antisecretory factor (FIG. 2). Coomassie
brilliant blue staining of the proteins revealed discrete bands for
each protein except for the GST-AF-1 protein which manifested
degradation into smaller components.
[0069] Solid phase peptide synthesis
[0070] Smaller peptides (P.sub.7 to P.sub.18 in Table 1) was
produced (K. J. Ross-Petersen AS) on solid phase in an Applied
Biosystems peptide synthesizer. The purity of each peptide was
93-100% as evaluated on reversed phase HPLC on Deltapak C18, 300 A
using a linear gradient of 0.1 % trifluoro acetic acid in
water/acetonitril.
[0071] Amino acid sequencing
[0072] Protein sequence analysis was performed to further validate
the identified ORF. The pure AF proteins were run in 10% macro-slab
gel SDS-PAGE (14) and the proteins transferred to a Problot
membrane (Applied Biosystems) by electroblatting (Bio-Rad). Spots,
visualized by Ponceau S staining, were excised from the blot and
the first 20 amino acids of the proteins were sequenced by
automated Edman degradation on an automatic sequencer (Applied
Biosystems).
[0073] The N-terminal sequences of clone-2 and clone-3 were
determined, and shown to perfectly match amino acids 63-75 and
130-140, respectively, of the predicted sequence (FIG. 1, SEQ ID
NO:2).
[0074] Comparison with other protein sequences available from
GenBank revealed that the sequence of rAF (FIG. 1, SEQ ID NO:2) is
unique in all its parts and no similar sequence has been
reported.
[0075] The first ten residues of the protein appear to be
relatively hydrophobic when analyzed according to KyteDoolittle
(22) and might constitute a signal peptide, which is cleaved out
prior to exocytosis of the protein. This interpretation is
supported by the Western blot analyzes (FIG. 3) in which the
recombinant protein appeared to have a slightly higher molecular
mass than the protein extract from pituitary gland. Some of this
difference, however, might also be due to the additional five amino
acids in the recombinant protein constituting the trombin cleavage
site of the fusion protein.
EXAMPLE 4
Production and Testing Antisera Against rAF
[0076] Antisera Against Recombinant GST-AF Fusion Protein
[0077] Antibodies against the purified fusion proteins GST-AF-1,
GST-AF-2 and thrombin-cleaved pure AF-I protein (=rAF) for use in
ELISA, Western blot and immunohistochemical studies were produced
in rabbits. Each rabbit was given 100 pg of antigen In I ml PBS
mixed with an equal volume of Freund's complete adjuvant; each
immunization was distributed in 8-10 portions injected in the back
intracutaneously. Two booster dozes with 50 .mu.g antigen were
injected at 3 and 5 weeks, the last one without Freund's complete
adjuvant. The rabbits were bled 6 days after last booster and sera
were prepared and stored at -20.degree. C. The sensitivity of the
antiserum was tested with a dot blot assay. GST-AF-2 was applied on
an ECL nitrocellulose membrane in 1/5 dilutions, and the antiserum
diluted 1:1000. The membrane was blocked with 1% bovine serum
albumin (BSA) in PBS at 4.degree. C. for 16 h, and then incubated
for 11/2 h with a 1:800 dilution of rabbit anti-GST-AF or porcine
AF antiserum. The blot was developed with alkaline
phosphatese-conjugated goat anti-rabbit immunoglobulin followed by
5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium
(Boehringer Mannheim). The estimated limit for antigen detection
was about 1 ng in this test.
[0078] SDS-polyacrylamlde gel electrophoresis and
immunoblotting
[0079] SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of human
and porcine pituitary gland extracts and pure AF-proteins was
performed in 10% acrylamide minislab gels, essentially as described
by Laemmli (4) with the modification that bis-acrylamide as a
cross-linker was replaced by N,N'-diallyltartardiamide with the
corresponding molarity. Pyronin Y (Sigma) was used as a marker of
the electrophoretic front. Prestained molecular weight reference
were purchased from BDH. Proteins were then either stained with
Coomassie brilliant blue or transferred electrophoretically to 0.45
mm pore-size ECL nitrocellulose (Amershem) for immunoblotting. The
subsequent incubations with BSA, conjugated anti-IgG and alkaline
phosphatase substrate were the same as for the dot blot assay
described above.
[0080] As stated above Coomassie Brilliant Blue staining revealed
no discrete band for the GST-AF-1 protein, which was probably due
to proteolytic degradation into smaller components. However, in the
Western blot analyzes the full length protein gave a much stronger
signal than the degradated products (FIG. 2b). The strong reaction
with the antiserum against porcine AF indicated that the
recombinant proteins indeed have the same immunoreactivity as AF.
The molecular weight of the full length protein appeared to be
about 60 kDa which is higher than the true mol. wt of 41139 Da
estimated from the amino acid composition. Furthermore, the
proteins were also immunoblotted and probed with antiserum raised
against GST-AF-2, which bound to the thrombin-cleaved proteins
(FIG. 3).
[0081] Antiserum against recombinant GST-AF-2 reacted with the
naturally occurring AF protein of an apparent mol mass of 60 kDa,
and with some smaller components, probably enzymatic degradation
products (FIG. 3a).
[0082] ELISA for determination of AP-concentrations
[0083] ELISA assays were performed using anti-AF-1 and anti-AF-2
according to a previously described method (5). As shown in FIG. 3b
the sensitivity of the test with the crude antiserum was between
1-10 .mu.g protein whereas the test with the affinity purified
antibody had a sensitivity between 5 and 50 ng protein,
EXAMPLE 5
Northern Blot Analysis of RNA from Pituitary Gland
[0084] Northern Blot Analysis
[0085] Human pituitary glands were obtained postmortem from
Sahlgrenska Hospital (permission given by Swedish Health and
Welfare Board; 2% transplantationslagen, 1975:190). To obtain RNA,
pituitary glands were extracted with guanidinium thiocyanate RNA
according to Chomczynski and Sacchi (6). Polyadenylated RNA was
selected by means of a commercial kit (Pharmacia) using columns
with oligodT-cellulose. In addition, a pool of human pituitary mRNA
from 107 individuals purchased from Clontach was used. Five .mu.g
of each sample of poly(A+)RNA was glyoxal-treated and
electrophoresed in a 1.2% agarose gel (7). After capillary alkaline
transfer for 3 h in 0.05 M NaOH to Hybond N+nylon membranes
(Amersham), prehybridization and hybridization were carried out for
24 h each at 42.degree. C. The hybridization solution contained 50%
formamide, 5.times.SSPE, 10.times.Denhard's solution with 250
.mu.g/ml denaturated low-MW DNA and 50 .mu.g/ml polyadenylic acid.
The blots were probed with four different antisense 28 bp
oligonuclaotides comprising the positions 132-105 (primer E),
297-270 (primer F), 748-721 (primer G) and 833-806 (primer H) of
the sequence (FIG. 1); the probes were 3'-end labelled with
terminal transferase (Boehringer Mannheim) plus
[.alpha..sup.32p]ddATP (Amersham) and purified on Nick columns
(Pharmacia). Five postwashes in 5.times.SSPE/0.1%
SDS-0.5.times.SSPE/0.1% SDS were made at 42.degree. C. for 30 min
each time, with a repeat of the last wash. Filters were exposed to
Hyperfilm MP (Amersham) for 7 days.
[0086] Expression in pituitary gland
[0087] Northern blot analyzes were performed with a mixture of four
oligonucleotide probes hybridizing with different sequences along
the cloned cDNA (FIG. 4). The probes hybridized with a single band
of about 1400 bp in the separated mRNA from pituitary gland. The
strongest signals were obtained with the human material, but the
porcine material also cross-reacted.
EXAMPLE 6
Distribution of AF in Sections of Pituitary Gland
[0088] Species and Tissues
[0089] Human pituitary glands were obtained postmortem from
Sahlgrenska Hospital (permission given by the Swedish Health and
Welfare Board; .sctn.2 transplantationslagen, 1975:190). Glands
were kept frozen at -70.degree. C., except those used for
histological examination which were fixed for 24 h in 4%
paraformaldehyde dissolved in phosphate-buffered saline (PBS=0.15 M
NaCl, 0.05 M sodium phosphate, pH 7.2) and thereafter transferred
to 7.5% sucrose in PBS. Pituitary glands from pigs, 5-7 months old,
obtained from a slaughter house, were placed on dry ice during
transport and kept frozen at -70.degree. C. until used.
Sprague-Dawley rate, 2-3 months old, were obtained for bioassay
from B & K Universal AB, Sollentuna, Sweden. Rabbits (New
Zealand White) for immunizations were obtained from Lidk{haeck over
(o)}ping Kaninfarm, Sweden.
[0090] Immunohistochemistry
[0091] The fixed pituitary glands were frozen in liquid nitrogen,
and cryo sections, 7 .mu.m thick, were prepared. From each sample
5-10 sections comprising different parts of the gland were fastened
to microscope slides. The sections were blocked in 5% fat-free
dried milk and incubated with primary rabbit antiserum
(anti-GST-AF-2 fusion protein) diluted 1:4000-1:8000 in a humid
chamber overnight at 4.degree. C. After rinsing in buffer, the
specimens were incubated for 1 h at 23.degree. C. with alkaline
phosphatase-conjugated swine anti-rabbit immunoglobulins diluted
1:50 (Dako A/S). The immunoreaction was visualized with phosphatase
substrates as described elsewhere (8). Control sections were
incubated with immune serum absorbed with an excess of OST-AF-2
protein or with all incubation steps except the primary
antibody.
[0092] Distribution of AF in sections of pituitary gland.
[0093] The distribution of AF in sections of human pituitary glands
was studied with immunohistochemical techniques (FIG. 5). In all
specimens investigated, a moderate number of cells in the
adenohypophysis were stained; the immunostained material appeared
to be located in granules in the cytoplasm; preabsorption of the
immune serum with an excess of GST-AF-2 protein abolished the
signal. No staining was observed in the posterior part
(neurohypophysis).
[0094] The distribution of immunoreactive material in the pituitary
gland demonstrated solely intracellular distribution of AF in
secreting cells of the anterior lobe (adenophypophysis). The
proteins emanating from this lobe include growth hormone,
thyrotropin, corticotropin, prolactin and lutainising hormone. The
passage of these hormones from intracellular localization to the
vascular system is triggered by releasing factors produced by
neuroandocrinic cells in the hypothalamus.
EXAMPLE 7
Biological Activity of rAF
[0095] Antisecretory Activity
[0096] The antisecretory activity was measured in a rat intestinal
loop model previously described (9). A jejunal loop was challenged
with 3 .mu.g of cholera toxin. Either different doses of purified
AF-1-proteins or PBS (control) was injected before or after the
challenge with cholera toxin. The weight of the accumulated fluid
in the intestinal loop (mg/cm) was recorded after five hours. Each
AF preparation was tested in at least six rats. Fisher's PLSD was
used for statistical analysis of the data.
[0097] Biological activity of rAF protein
[0098] The biological activity of the pure rAF protein of clone-1
produced in E. coli was tested in a rat model. The capacity of the
rAF to inhibit intestinal fluid secretion when injected
intravenously 20-30 sec before intestinal challenge with cholera
toxin is shown in FIG. 6. In control animals injected with buffer
only, the cholera toxin caused a pronounced secretion, 412.+-.9 mg
fluid per cm intestine. The pure rAF caused dose-dependent
inhibition of the cholera secretion which was significantly
different from the response to the buffer (p<0.01, n=6). Nine ng
of clone-1 protein is sufficient to reduce the response by 34%,
whereas 44 ng (10.sup.-12 mol) and 220 ng reduced it by 46% and
78%, respectively. The biological activity of recombinant AF is
greater than that of any enterotoxin known to us and greater than
that of any intestinal hormone or neuropeptide modifying water and
electrolyte transport. Moreover, the level of activity of human rAF
in rat is surprisingly high which probably reflects a ubiquitous
structure conserved in rAF molecules from different species. This
hypothesis is supported by the cross-reactivity between human and
porcine material obtained in the Western blot and Northern blot
analyzes.
[0099] The capacity of 0.5 .mu.g of rAF to inhibit intestinal
secretion when injected intravenously 20-30 sec before and 90 min
after cholera toxin challenge was compared (FIG. 7). Both
administrations gave significant inhibition compared to control
animals (p<0.01, n=6). Thus, in contrast to natural AF, the
recombinant protein was also efficient when given after toxin
challenge which make rAF useful for therapeutic treatment of
diarrhea.
[0100] 3 .mu.g rAF was injected in a 8-10 cm long loop placed
immediately proximal to the loop which was challenged with cholera
toxin. The rAF was either induced 20-30 sec before or 90 min after
the toxin-challenge. In. FIG. 8 it is shown that both test groups
obtained a significant reduction of the fluid secretion compared to
controls (p<0.01, n=6); no difference was observed between the
two test groups. This experiment suggests that rAF is active after
oral administration and might be used as an additive in animal feed
provided that no serious side effect is obtained.
[0101] In the Examples described above, the rAF was produced in
Epicurian Coli XL-1 cells. In these cells much of the produced rAF
was degradated into smaller peptides. When rAF was produced in BL21
cells only a small portion of the rAF was degradated while in Top 1
cells no degradation was observed. Surprisingly the biological
activity was proportional to the extent of degradation, i.e. more
degradation resulted in higher activity. Therefore various shorter
fragments were produced in order to test for their possible
biological activity.
[0102] As shown in Table 1, these fragments were tested
intravenously prior to cholera toxin challenge in the same way as
described above for the intact rAF. The peptides expressed by clone
2 and 3 tested in amounts of 0.1, 1 and 10 .mu.g had no effect on
the toxin response. In contrast one microgram of the peptide
expressed by the RACE fragment (clone 4) had a pronounced effect. A
lot of shorter constructs were made from the RACE fragment and
expressed in pGex-1-lambda. As shown in Table 1, the active site
was found to be situated between amino acid residue 35 to 51. In
order to determine more exactly the active site three small
peptides were made by solid phase peptide synthesis. Two of them
were active, peptide 35-46 (P3) and peptide 35-42 (P1); the latter
octapeptide IVCHSKTP, (P1) was active in a dose less than 1 ng
being almost as active in a molar basis as the intact rAF. In
contrast a shorter hexapeptide VCHSKT (P2) exerted no effect when
tested in doses between 1 ng and 10 .mu.g.
[0103] A peptide X.sub.1VCX.sub.2X.sub.3KX.sub.4R corresponding to
the human fragment P1 but with certain changes and/or deletions,
have also been produced by site directed mutagenesis and tested for
biological activity. Comparison was also made of sequences from
bovine and porcine cDNA. These studies suggested the following
changes and/or deletions:
[0104] X.sub.1 is I or none
[0105] X.sub.2 is H, R or K
[0106] X.sub.3 is S, L or another neutral amino acid
[0107] X.sub.4 is T or A.
1 TABLE 1 Inhibition of cholera secretion*** Code Oligonucleotide*
Peptide** pmol ED50 N1 63-301 1-80 + 4 N2 168-301 36-80 + 6 N3
168-215 36-51 + 3 N4 122-170 21-36 - N5 186-269 42-69 - P3
S.P.S.*** 35-46 + 7 P1 S.P.S. 35-42 + 5 P2 S.P.S. 36-41 - *Position
of the basepair in (SEQ ID NO: 1) which was expressed in the
construct made from the AF cDNA and pGex-1-lambda. **Positions of
the amino acid-residues (SEQ ID NO: 2) in AF at the start and end
of the synthesized peptide. ***Inhibition of cholera toxin induced
fluid secretion in a ligated rat intestinal loop; the amount (pmol)
causing halfmaximal inhibition (ED50) is noted for active peptides.
****These peptides were produced by solid phase synthesis.
[0108] The effect of rAF on inflammation in the intestinal mucosa
was also tested in the rat intestinal loop model. Thus, 20 rate
were challenged with 0.5 .mu.g of toxin A from Clostridium
difficile (10) and the inflammatory and fluid secretion measured
after 2.5 and 5 hours, respectively (10+10 rats). Half of the rats
in each group received 100 ng of rAF intravenously 30 sec prior to
the challenge; the other half received PBS buffer as control. After
killing the rat, the loops was dissected out, and the middle 2-3 cm
part of the loops were frozen on dry ice. The frozen specimens were
then sectioned in 8 .mu.m thick sections by use of a Leica
cryostat. The sections were stained to demonstrate alkaline
phosphatases by enzyme histochemistry. Alkaline phosphatases are
expressed by the intestinal epithelial cells and the staining
allows an assessment and of the integrity of the intestinal
epithelium.
[0109] The results revealed (FIG. 9) that the control rats
developed extensive damage of the intestinal mucosa: after 2.5 h
shedding of epithelial calls from the basal membrane was observed
together with necrotic tissue, whereas extensive bleeding was
observed after 5 h. In contrast, animal treated with rAF developed
no shedding, necrosis or bleeding. The toxin A-induced fluid
secretion was also inhibited from 199.+-.4 to 137.+-.5 mg/cm after
2.5 h (p<0.01) and from 421.+-.3 to 203.+-.6 mg/cm after 6 h (5
rats/group, p<0.01).
[0110] A similar experiment was performed with 0.5 .mu.g of the
peptide IVCHSKTR (=P1) replacing the rAF protein. The octapeptide
achieved the same effect on toxin A-induced intestinal inflammation
and fluid secretion as shown in FIG. 9.
[0111] Toxicity
[0112] In order to test the toxicity of rAF it was injected in a
high dose, 50 .mu.g per rat. No obvious toxic reaction was
registered during an observation period of one week.
EXAMPLE 8
Biological Activity of rAF on Intestinal Permeability
[0113] In order to evaluate the effect of rAF on the permeability
of an organic substance dissolved in the blood a test with Evans
blue dye was performed according to a previously described method
(11). The experiment was initially performed as described above in
Example 7 and FIG. 5 with intravenous injection of rAF prior to
cholera toxin challenge. However, no fluid secretion was measured
but 90 min after toxin challenge Evans blue dye, 1 ml of a 1.5%
solution in PBS, was injected intravenously. The dye was allowed to
circulate for a 5 min long period. Thereafter the rat was subjected
to transcardial perfusion via the left ventricle-right atrium
(using a peristaltic pump [Cole Parmer Instruments, Chicago, Ill.,
USA]) with 200 ml of 4.degree. C. PBS/Alsevier's (1/1 ratio)
solution during a period of some 150 sea, performed under ether
anaesthesia. This procedure was undertaken in order to remove all
of the EB present in the vascular system, leaving only the EB in
the interstitial tissue to be detected by the formamide extraction
of the dye.
[0114] The results in Table 2 demonstrate that CT-challenge
significantly (p<0.001) increases the amount of EB that can be
extracted from the intestinal tissue with some 43%, while an
intravenous injection of 1 BrT prior to cholera toxin challenge
prevent this increase, i.e. the amount of EB extracted from the
tissue in group 1 (control) did not differ from that in group 3 (1
rAF+CT)
2TABLE 2 EB/g % increase of Group Challenge ng int. tissue .times.
10.sup.-07 EB-kono 1 PBS + PBS 6 29.3 .+-. 1.0 - 2 PBS + CT 6 51.8
.+-. 1.3 43 (p < 0.001) 3 1rAF + CT 6 29.6 .+-. 1.5 0 NS
[0115] The results shown in rigs 10 and 11 demonstrate the
extravasation of the azo dye Evans blue in the small intestine and
in the corresponding plexus choroideus from the lateral ventricles
of the brain after intestinal challenge with cholera toxin, with
and without previous treatment of the rats with P1 (IVCHSKTR),
[0116] The experiments were performed in the following way: Male
Sprague-Dawley rats, weighing 350 g, were starved for 18 h prior to
the experimental procedure, but had free excess to water. The rats
were used in groups of six. The peptide P1, cholera toxin (CT), and
PBS were administrated according to Table 3.
3 TABLE 3 Group iv inj. 1* po. inj.* iv. inj. 2* A P1 CT EB B PBS
CT EB C PBS PBS EB *P1 (iv.) injection 1 were given in a volume of
2 ml PBS, the peroral (po.) injection were given in a volume of 5
ml, the intravenous injection 2 consisted of 1.5 ml of 3% Evans
blue dissolved in PBS. Ether was used for anaesthesia during the
performance of all injections.
[0117] The i.v. injection of P1 (0.5 .mu.g) or of PBS were
performed 10-15 sec before the peroral challenge with 100 .mu.g CT
or with PBS; 60 min after the peroral challenge, the rats were
anaesthetized with ether and injected iv. with Evans blue. The dye
was allowed to equilibrate for another 30 min, whereafter the rats
were again anaesthetized with ether and perfused intracardially via
the left ventricle with 250 ml of Alsevers solution/PBS=50/50, in
order to remove all dye present in the vascular system. After this
perfuming treatment, performed during some 2-3 min, the
fluorescence registrated should represent dye present only outside
the vascular system.
[0118] The brain and a part of the small intestine were sampled and
frozen on dry ice and cryostat sections, 8 .mu.m thick, were
prepared. The sections were air-dried and mounted in a
xylene-containing mounting media. The sections were viewed in a
Zeiss fluorescence microscope using a filter combination identical
to that used for rhodamin-emitted fluorescence.
[0119] The results in FIGS. 10 and 11 demonstrate that the
fluorescent intensity (white color) is of a similar magnitude in
both the small intestine (FIG. 10) and in the plexus choroideus
(FIG. 11) in group A (P1 iv+CT po) and C(PBS iv+PBS po). Compared
to the high fluorescent intensity in the small intestine as well as
in the plexus choroideus in group B (PBS iv+CT po), the results
clearly demonstrate that injection of the octapeptide prior to
toxin challenge inhibits the CT-induced extravascular penetration
of Evans blue. The results suggest that this holds true not only in
the vascular system of the small intestine, but also in the plexus
choroideus of the lateral ventricles of the brain.
[0120] In conclusion: the effect of intravenous octapeptide
IVCHSKTR administration inhibits cholera toxin-induced
extravascular penetration of Evans blue in the small intestine as
well as in the plexus choroideus in the central nervous system.
Thus, the action of rAF and its peptide derivatives is not confined
to the small intestine only, but influences also the permeability
of blood vessels in the central nervous system. These findings
indicate that rAF and its peptide derivatives can be used to
reverse pathological intracranial pressure, pressure alteration in
the middle ear and various forms of permeability changes in blood
vessels.
References
[0121] [1] Young, R. A. and Davis, R. W. (1983) Proc. Natl. Acad.
Sci. USA. 80, 1194-1198
[0122] [2] Sambrook., J., Fritsch, E. F., Manistis, T. (1989)
Molecular Cloning: A Laboratory Manual, pp 1.74-1.84, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0123] [3] Frohman, M. A., Dush, M. K., and Martin, G. R. (1988)
Proc. Natl. Acad. Sci., USA 86, 8998-9002.
[0124] [4] Laemmli, U. K. (1970) Nature 227,680-685.
[0125] [5] Zachrisson, G., Lagarg.ang.rd, T. and L{haeck over
(o)}nnroth, I. (1986) Acta Path. Microbial. Immunol. Scand. C, 94,
227-231.
[0126] [6] Chomczynaki, P., Sacchi, N. (1987) Analyt. Biochem. 162,
156-159.
[0127] [7] Sambrook, J., Fritsch, E. F., Maniatis, T. (1989)
Molecular Cloning: A Laboratory Manual, pp 7.40-7.42, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0128] [8] Jennische, E., Matejka, G. L. (1992) Acta. Physiol.
Scand., 146,79-86.
[0129] [9] Lange, S. (1982) FEMS Microbiol. Lett. 15, 239-242.
[0130] [10] Torres, J. F., Jennische, E., Lange, S. and L{haeck
over (o)}nnroth, I. (1990) Gut 781-785
[0131] [11] Lange, S., Delbro D S, Jannische E. Evans Blue
permeation of intestinal mucosa in the rat. Scand. J.
Gastroenterol. 1994, 29:38-46.
Figure Legends
[0132] FIG. 1a and continued on FIG. 1b. Nucleic acid sequence (SEQ
ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2) of the new
human protein. The confirmed amino acid sequence is underlined.
Fig. 1c. Horizontal map showing cloned cDNA and oligonucleotide
primers.
[0133] FIG. 2. Coomassie brilliant blue-stained SDS-polyacrylamide
minlgel (A) and immunoblot probed with antisera against porcine AF
(B). Lanes with unprimed numbers contain
glutathione-agarose-purified GST-AF fusion proteins AF-1, AF-2 and
AF-3, whereas lanes with primed numbers contain the fusion proteins
cleaved with thrombin. Molecular weight references (R), (BDH), are
indicated on the left. The GST-AF-1 fusion protein is highly
degraded but the immunoblot analysis shows only the detection of a
full-length protein and spontaneous thrombin cleavage product.
There is a 26 kDa product in the GST-AF-3 protein, probably the
glutathione S-transferase-tail that has been independently
expressed.
[0134] FIG. 3a. Western blot using antiserum against recombinant
protein AF-2. To the left, porcine (P) and three human (H1, H2, H3)
pituitary glands; and to the right, the three recombinant proteins
AF-1, AF-2 and AF-3 (see FIG. 2) were applied; in the center the
molecular weight standard (R).
[0135] FIG. 3b. Enzyme linked immuno-assay (ELISA) of rAF using
crude antiserum and affinity purified antibodies raised in
rabbit.
[0136] FIG. 4. Autoradiogram of Northern blots of RNA from a human
and porcine pituitary gland (p=pooled and i=individual material).
Five .mu.g of purified mRNA was applied in each basin; 3'-end
.sup.32P-labelled oligonucleotide probes were used and the
autoradiogram developed after 7 days,
[0137] FIG. 5 Cryosections of adenohypophysis stained with
antiserum against recombinant protein GST-AF-2. A. Sections
incubated with immune serum showing scattered cells with varying
degrees of positive immunoreactivity (solid arrows). Many calls
completely lack staining (open arrows). B. Serial sections to A
incubated with immune serum preabsorbed with excess of recombinant
protein GSTAF-2, There is no specific staining of the cells. C and
D. Larger magnifications of immunopositive cells demonstrating
cytoplasmatic staining of the endocrine cells, n=nucleus,
c=cytoplasms.
[0138] FIG. 6. Biological activity of recombinant protein AF-1
testing inhibition of cholera toxin-induced fluid secretion. Graded
doses of the protein were injected intravenously in rat; three
.mu.g of cholera toxin was injected into an intestinal loop; after
five hours the accumulated fluid (mg/cm intestine) in the loop was
measured. Each value represents the mean .+-.S.A.E. of a group of
six animals.
[0139] FIG. 7. Biological activity of intravenously injected rAF-1;
0.5 .mu.g of rAF was administrated 20-30 sec before or 90 min after
challenge with 3 .mu.g of cholera toxin in an intestinal loop of
rat.
[0140] FIG. 8. Biological activity of intraluminarly injected
rAF-1; 3 .mu.g of rAF was injected 20-30 sec before or 90 min after
challenge with 3 .mu.g of cholera toxin in an intestinal loop of
rat; the rAF was injected about 5 cm proximate to the loop in which
the toxin was injected.
[0141] FIG. 9 A (.times.2.5) is control (PBS) loops showing
cellular debris in the intestinal lumen (L), but no staining of the
remaining mucosa, which suggests a total destruction of the
epithelial lining. B (0.5 .mu.l of P1 prior to toxin challenge)
shows a clearly delineated epithelial lining forming villi,
suggesting a conserved and normal intestinal mucosa. L=intestinal
lumen. Bars=500 .mu.m. C (.times.10) shows the destructed mucosa in
the PBS-treated control group, and D shows the corresponding mucosa
in the experimental (P1-treated) group. The black arrow point at
the epithelial lining, LP=lamina propria, mm=muscularis mucosa,
open arrow point at the crypt cells. Bars=100 .mu.m. E (.times.25)
shows the destructed mucosa in the control (PBS-treated) group, and
F shows a corresponding magnification from a rat subjected to P1
treatment prior to toxin challenge. Bars=50 .mu.m.
[0142] FIG. 10. Evans blue fluorescence in jejunal specimens from
three groups of rats treated with cholera toxin (CT) or control
buffer (PBS); pretreatment with antisecretory peptide P1 or control
buffer (PBS). LP=lamina propria. Black arrow indicating epithelial
cell lining; open arrow head indicating crypt cells. Bars=100
.mu.m.
[0143] FIG. 11. Evans blue fluorescence in plexus choroideus
specimens from the rats shown in FIG. 10. Bars=50 .mu.m.
Sequence CWU 0
0
* * * * *