U.S. patent application number 12/807237 was filed with the patent office on 2011-05-26 for pdja1, a cardiac specific gene, corresponding proteins, and uses thereof.
This patent application is currently assigned to University of Medicine and Dentistry of New Jersey. Invention is credited to Christophe Depre, Stephen F. Vatner.
Application Number | 20110124714 12/807237 |
Document ID | / |
Family ID | 30118209 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110124714 |
Kind Code |
A1 |
Depre; Christophe ; et
al. |
May 26, 2011 |
pDJA1, a cardiac specific gene, corresponding proteins, and uses
thereof
Abstract
The present invention provides novel nucleic acid and protein
sequences for methods and compositions for treating, screening, and
diagnosing cardiovascular disease and methods for using these genes
and gene products for prevention of cardiac cell death and
prevention of cardiac tissue damage resulting from ischemic events
in cardiac tissue, as well as other tissue that is subject to
damage resulting from an ischemic event. The genes, gene products
and agents of the invention are also useful for treating other
related clinical or coronary events such as angina, myocardial
infarct (MI), and stroke, for monitoring the effectiveness of their
treatment, and for drug development. The genes, gene products and
agents of the present invention are also provided as pharmaceutical
compositions for treatment of cardiovascular disease, ischemic
heart disease, myocardial infarct and related conditions. Kits are
also provided for the diagnosis, treatment and prognosis of cardiac
diseases and related conditions.
Inventors: |
Depre; Christophe; (New
York, NY) ; Vatner; Stephen F.; (New York,
NY) |
Assignee: |
University of Medicine and
Dentistry of New Jersey
|
Family ID: |
30118209 |
Appl. No.: |
12/807237 |
Filed: |
August 31, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11365484 |
Mar 1, 2006 |
7803908 |
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12807237 |
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10429223 |
May 2, 2003 |
7009038 |
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11365484 |
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60377578 |
May 2, 2002 |
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Current U.S.
Class: |
514/44R ;
536/23.5 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101; C07K 14/47 20130101; C12Q 2600/106
20130101; C07H 21/04 20130101; C12Q 2600/156 20130101; A61P 9/00
20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/44.R ;
536/23.5 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C07H 21/00 20060101 C07H021/00; C07H 21/04 20060101
C07H021/04; A61P 9/00 20060101 A61P009/00; A61P 9/10 20060101
A61P009/10 |
Goverment Interests
GOVERNMENT RIGHTS CLAUSE
[0002] The research leading to the present invention was supported,
at least in part, by National Institutes of Health grants HL33065,
PO1 HL 59139, PO1 HL 69020, AG 14121 and HL 33107 and AHA Scientist
Development Grant 0230017N. Accordingly, the Government may have
certain rights in the invention.
Claims
1. An isolated nucleic acid, comprising a pDJA1 coding
sequence.
2. The nucleic acid of claim 1, wherein the nucleic acid comprises
the sequence of SEQ ID NO: 1, and recombinant DNA molecules, cloned
genes, degenerate variants, mutants, analogs, or fragments
thereof.
3-4. (canceled)
5. A biomarker associated with and/or predictive of cardiovascular
disease, wherein said cardiovascular disease is selected from the
group consisting of atherosclerosis, coronary heart disease,
ischemic heart disease, myocardial infarction, angina, stroke and
other related conditions related to or resulting from an ischemic
event, comprising the nucleic acid of SEQ ID NO:1.
6-17. (canceled)
18. A pharmaceutical composition comprising a therapeutically
effective amount of pDJA1 nucleic acid and a pharmaceutically
acceptable carrier.
19. (canceled)
20. A pharmaceutical composition comprising a therapeutically
effective amount of an agent that upregulates the expression and/or
activity of the isolated nucleic acid of claim 1 and a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application, which
claims priority to copending non-provisional U.S. Ser. No.
10/429,223, filed on May 2, 2003, which is a non-provisional
application claiming the priority of provisional U.S. Ser. No.
60/377,578, filed on May 2, 2002, the disclosures all of which are
hereby incorporated by reference herein in their entireties.
Applicants claim the benefits of these applications under 35 U.S.C.
.sctn.119(e) and 35 U.S.C. .sctn.120.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of cardiology
and the identification of genes and gene products involved in
protection of cardiac tissue against irreversible ischemic damage.
More particularly, the present invention relates to methods of
identifying and cloning novel cardioprotective genes, expressing
the gene products, and methods of using the genes or gene products
for prevention or treatment of damage to heart tissue arising from
ischemic events. Methods for diagnosing ischemic cardiac events are
also envisioned by use of the genes or gene, products of the
present invention. Methods of using a nucleic acid and/or a
protein, expressed in cardiac cells, to treat or prevent heart
damage and antibodies against the protein, to diagnose heart
damage, are provided for by the present invention. The instant
invention also provides compositions comprising, and methods of
using products of a novel gene designated pDJA1 and associated
variants thereof. Such gene products, as well as their binding
partners, agonists, and antibodies to the gene products can be used
for the prevention, diagnosis, prognosis and treatment of
cardiovascular disease.
BACKGROUND OF THE INVENTION
[0004] Cardiovascular disease, including, but not limited to,
atherosclerosis, ischemia, reperfusion, hypertension, restenosis
and arterial inflammation, is a major health risk throughout the
world. Ischemia is a condition wherein there is a lack of oxygen
supply in tissues or organs due to inadequate perfusion due to
atherosclerosis or restenotic lesions, stroke, or anemia, to name a
few. The most common cause of ischemia in the heart is
atherosclerotic disease of the coronary arteries. Myocardial
ischemia can also occur if myocardial oxygen demands are abnormally
increased, due to hypertension or aortic stenosis.
[0005] One of the most important therapeutic targets in the
treatment of cardiovascular disease has been the protection of
ischemic myocardium from necrosis. This has been a major focus for
basic and applied research over the past 30 years. More recently,
mechanisms of programmed cardiac cell death (apoptosis) have also
been studied extensively. Both necrosis and apoptosis result in the
irreversible loss of contractile performance. An unexplored
corollary to protection from cell death is the enhancement of cell
survival.
[0006] Heat shock proteins are involved in the folding, degradation
and translocation of intracellular proteins (Benjamin I, et al.,
Stress (heat shock) proteins, (1998), Circ Res. 83: 117-132), but
they also participate in the protection against apoptosis and in
cell growth (Mehlen P. et al, Small stress proteins as novel
regulators of apoptosis. Heat shock protein 27 blocks FAS/APO-1 and
staurosporine-induced cell death. J Biol. Chem. (1996); 271:
16510-16517; Beere H., et al, Heat-shock protein 70 inhibits
apoptosis by preventing the recruitment of procaspase-9 to the
Apaf-1 apoptosome, Nature Cell Bio., (2000); 2: 469-475; Li, C. et
al., heat shock protein 70 inhibits apoptosis downstream of
cytochrome c release and upstream of caspase-3 activation, J Biol.
Chem., (2000); 275:25665-26571; Kamradt, M., et al., The small
heat-shock protein .alpha.B-crystallin negatively regulates
cytochrome c- and caspase-8-dependent activation of caspase-3 by
inhibiting its autoproteolytic maturation, J Biol. Chem., (2001);
276: 16059-16063). They are crucial effectors of the program of
cell survival, which protects cells against irreversible damage and
accelerates functional recovery after stress (Latchman, D., Heat
shock proteins and cardiac protection, Cardiovasc Res. (2001); 51:
637-646). Two main forms of heat-shock proteins in E. Coli, called
DnaK and DnaJ, have been conserved in eukaryotes (Kelley, W., How J
domains turn on Hsp70s., Cur Biol. (1999); 9: R305-R308). In
mammalian cells, the chaperone HSP40 is the homologue of DnaJ.
Several isoforms of DnaJ-like/HSP40 homologues have been cloned,
that differ by their tissue distribution and their protein
interactions. The role of these co-chaperones is to stimulate the
ATPase activity of the cognate HSP70 (Russell, R., et al., DnaJ
dramatically stimulates ATP hydrolysis by DnaK: insight into
targeting of Hsp70 proteins to polypeptide substrate, Biochemistry,
(1999); 38: 4165-4176; Minami, Y., et al., Regulation of the
heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog,
Hsp40, J Biol Chem., (1996); 271: 19617-19624) and to modulate its
substrate-binding capacity. The heat-shock response is particularly
developed in cardiac cells, which are long-lived, post-mitotic
cells submitted to high oxidative stress (Williams, R., et al.,
Protective responses in the ischemic myocardium, J Clin Invest.,
(2000); 106: 813-818). During ischemia/reperfusion, this response
is important to tilt the balance between cell survival and cell
death.
[0007] Myocardial stunning refers to a form of non-lethal, fully
reversible myocardial dysfunction that follows an acute episode of
ischemia (Heyndrickx, G R, et al., Regional myocardial functional
and electrophysiological alterations after brief coronary artery
occlusion in conscious dogs, J Clin Invest. (1975) 56: 978-985;
Kloner, R A., et al., Consequences of Brief Ischemia: Stunning,
Preconditioning, and Their Clinical Implications: Part 1,
Circulation, (2001); 104: 2981-2989). The syndrome of stunning is
prevalent in different etiologies of coronary artery disease,
including stable or unstable angina pectoris, myocardial
infarction, and post-surgical dysfunction (Bolli, R., et al.,
Molecular and cellular mechanisms of myocardial stunning, Physiol
Rev., (1999); 79: 609-634). Due to the major prevalence of ischemic
heart disease, stunning is of paramount importance because it
corresponds to a condition in which myocardial viability is
maintained. Unraveling the molecular mechanisms of cardioprotection
in stunned myocardium can open new avenues to salvage dysfunctional
cardiac tissue and prevent cardiac cell loss. Especially, a better
understanding of the mechanisms by which the molecular and cellular
adaptations maintain cell survival should open new therapeutic
opportunities.
[0008] It would, therefore, be beneficial to provide for specific
genes, gene products, compositions and methods for the treatment
and diagnosis of cardiac disease, including ischemic cardiac
events, and to provide methods that would identify individuals with
a predisposition for such conditions, and other types of
cardiovascular disease or related conditions, and hence are
appropriate subjects for preventive therapy.
SUMMARY OF THE INVENTION
[0009] It is well recognized that myocardial ischemia leads to cell
death, whether by necrosis or apoptosis, and that survival of
postischemic myocardium depends on factors that limit necrosis
and/or apoptosis. The present invention relates to the discovery
that ischemia, followed by reperfusion induces a gene program of
cell survival in cardiac tissue or other tissue exposed to an
ischemic event.
[0010] A first aspect of the invention provides for the
identification, expression and use of genes and gene products that
counteract apoptosis, thus acting as cytoprotectants and inducers
of cell growth. In a preferred embodiment, a novel gene, designated
pDJA1, the nucleic acid sequence of which is provided in SEQ ID NO:
1, and variants thereof, have an expression pattern that is
up-regulated in cardiac tissue and cardiac cell lines. The
invention relates to the use of said gene, gene products, and
agonists of said gene or gene products (pDJA1 and variants thereof,
cDNA, RNA, and/or protein, small synthetic organic molecules,
antibodies) as targets for diagnosis, drug screening and
development of therapies for cardiovascular disease. In a preferred
embodiment, the invention provides for methods of using the protein
encoded by said gene, provided herein as SEQ ID NO: 2, and variants
thereof, or nucleic acids that encode said proteins or variants
thereof for the treatment, prevention and diagnosis of
cardiovascular disease.
[0011] A second aspect of the invention provides for a biomarker
associated with and/or predictive of cardiovascular disease
including atherosclerosis, coronary heart disease and clinical and
coronary events including myocardial infarction, angina, stroke and
other related conditions related to or resulting from an ischemic
event (eg., an episode wherein tissue such as cardiac tissue is
deprived of oxygen for a period of time due to an occlusion, which
results in cell death or damage), followed by reperfusion. In a
preferred embodiment, the biomarker comprises the nucleic acid of
SEQ ID NO: 1. In yet another preferred embodiment, the biomarker
comprises the amino acid sequence of SEQ ID NO: 2. The genes
encoding this protein biomarker were identified using subtractive
hybridization of cardiac tissue in a swine model of transient
ischemia. This biomarker correlates with the areas of cardiac
tissue that exhibit recovery following the ischemic event and
reperfusion thereafter. It is envisioned that the preferred
biomarkers, including the nucleic acid of SEQ ID NO: 1 and the
polypeptide encoded by the pDJA1 gene identified in SEQ ID NO: 2,
may be of diagnostic or prognostic use in a clinical setting.
Assays detecting this gene, or variants thereof, the protein or
polypeptide or fragments or variants thereof, may be used to assess
overall recovery from the ischemic event or to monitor disease
progression, and response to therapy. Such assays will augment
existing diagnostic methodologies and allow identification and
monitoring of patients. They will also facilitate the development
of therapeutic agents directed at cardiovascular disease or related
conditions, while potentially highlighting new targets for such
intervention. In addition, these biomarkers may have predictive
value in other chronic/acute disease states in which contributing
factors or resulting events in common with cardiovascular disease
or atherosclerosis occur including for instance, but not limited to
stroke, Alzheimer's Disease, tissue repair and various inflammatory
conditions.
[0012] A third aspect of the invention provides methods and
compositions for treatment of cardiovascular and/or ischemic heart
disease, coronary artery disease and clinically related conditions,
and for screening and development of agents for treatment of such
conditions. Preferred embodiments include use of the genes or gene
products of the present invention (eg. SEQ ID NOS: 1 and 2) for
delivery to a mammal in need of such therapy. In a further
preferred embodiment, the mammal is a human subject.
[0013] A fourth aspect of the invention provides methods for
diagnosing cardiovascular and/or ischemic heart disease, myocardial
infarct, coronary artery disease and clinically related conditions.
One embodiment includes the use of the genes or gene products
identified by the methods described herein, including but not
limited to those genes and gene products identified through use of
subtractive hybridization assays in a large mammalian model of
cardiac ischemia/reperfusion. In a preferred embodiment, the gene
useful for diagnosis of a coronary artery disease, ischemic heart
disease, myocardial infarct, carotid artery disease and other
clinically related conditions includes the gene identified in the
nucleic acid of SEQ ID NO: 1. Another preferred embodiment includes
the use of the gene products identified by the methods described
herein, including but not limited to the gene product identified as
the protein of SEQ ID NO: 2.
[0014] A fifth aspect of the invention provides antibodies, e.g.,
monoclonal and polyclonal and chimeric and bispecific antibodies,
capable of immunospecific binding to a specific gene or gene
product or any portion or fragment thereof, particularly a gene or
gene product disclosed herein. These antibodies may be utilized for
diagnostic or therapeutic purposes.
[0015] A sixth aspect of the present invention provides
pharmaceutical compositions comprising a therapeutically effective
amount of the genes or gene products of the present invention,
including but not limited to the nucleic acid of SEQ ID NO: 1 or
the protein of SEQ ID NO: 2, with a pharmaceutically acceptable
carrier for delivery to an individual in need of such therapy.
Included in this aspect of the invention are agonists of the gene
or gene products identified herein. Such agonists may be small
synthetic organic molecules, proteins, peptides, polypeptides or
antibodies. A further embodiment comprises a therapeutically
effective amount of an agent that upregulates the expression and/or
activity of the pDJA1 gene or gene product and a pharmaceutically
acceptable carrier. The pharmaceutical compositions may be
delivered orally, intravenously, intramuscularly, subcutaneously.
intrathecally, intracranially. They may be in the form of tablets,
capsules, suspensions, suppositories or in liquid form suitable for
intravenous delivery.
[0016] A seventh aspect of the invention provides kits that may be
used in the above recited methods and that may comprise single or
multiple preparations, or antibodies, together with other reagents,
e.g., labels, substrates, if needed, and directions for use. The
kits may be used for diagnosis of disease, or may be assays for the
identification of new diagnostic and/or therapeutic agents, or to
identify new targets for therapeutic agents.
[0017] An eighth aspect of the invention provides methods of
screening for agents that upregulate the expression or the
activity, of the genes or gene products of the present
invention.
[0018] A ninth aspect of the invention provides methods of treating
cardiovascular disease, including, but not limited to, coronary
heart disease, ischemic heart disease, stroke, atherosclerosis and
related conditions, comprising administering to a subject a
therapeutically effective amount of an agent or drug that
upregulates the expression or activity of genes and gene products
that aid in prevention of cardiac cell death. An agent described
herein may be used alone or in conjunction with other therapeutic
regimens or drugs currently used in the treatment of patients
having such cardiac diseases or related conditions. Included in
this is the use of the agents of the present invention with
standard therapies such as angioplasty or concurrently with the use
of stents.
[0019] A tenth aspect of the present invention provides for
elicitation of a genomic profile promoting cell survival following
a myocardial ischemic event, which includes the up-regulation of
genes involved in prevention of apoptosis, in cytoprotection and in
promotion of cell growth. Furthermore, if a program of cell
survival can be stimulated in the ischemic heart, this represents a
novel and important therapeutic strategy for patients suffering
from or prone to developing cardiovascular disease, or prone to
further subsequent ischemic events.
[0020] An eleventh aspect of the invention provides for screening
and identification of genes that are upregulated by
ischemia/reperfusion. In one embodiment, screening is conducted in
a large mammalian model of myocardial stunning using cDNA
subtractive hybridization (Depre, C., et al., Gene program for
cardiac cell survival induced by transient ischemia in conscious
pig, Proc Nat'l Acad Sci. U.S.A., (2001); 98: 9336-9341). In
particular, the genomic profile of ischemic myocardium was examined
in a model that is most relevant to clinical conditions, i.e., a
swine model of transient ischemia. Although the majority of
investigations on myocardial ischemia are conducted in rodent
models, major differences exist between rodents and larger mammals
(differences in heart rate, action potential, and calcium handling)
(Benjamin I., Circ. Res., (1998), 83:117-132; Mehlen, P. et al. J.
Biol. Chem. (1996), 271: 16510-16517). Thus, the best experimental
model to elicit a program of cell survival should include a
transient episode of ischemia reperfusion without irreversible
damage. This model induces myocardial stunning, which may be one of
the most frequently encountered sequelae of ischemia in patients
with ischemic heart disease (Beere, H. et al. Nature Cell Biol.
(2000) 2:469-475). Stunning is the prolonged dysfunction of the
ischemic heart that persists after reperfusion despite the
normalization of blood flow and that eventually resolves with
complete contractile recovery, provided no other ischemic episode
intervenes (Li, C. et al. J. Biol. Chem. (2000) 275: 25665-25671);
Kamradt, M. et al. J. Biol. Chem. (2001), 276: 16059-16063). The
activation of a program of cell survival would explain both the
full reversibility of dysfunction in stunned myocardium and the
protection against further ischemia, referred to as preconditioning
(Latchman et al., Cardiovacs. Res. (2001), 51: 637-646; Kelley W.
et al. Curr. Biol. (1999), 9:R305-R308). The genomic response
observed parallels the time course of myocardial stunning and
differs transmurally, related to the transmural differences in
reduction of blood flow during ischemia. One embodiment provides
for the identification of genes not previously characterized in
myocardium, wherein said genes are identified in a model of
transient ischemia followed by prolonged stunning, which elicits a
genomic profile of cell survival. The genes that are upregulated in
ischemic myocardium encode transcripts that are involved in
protective mechanisms against irreversible ischemic damage. A
preferred embodiment of the invention provides for a full-length
sequence and characterization of an isolated nucleic acid,
comprising a pDJA1 protein coding sequence. This nucleic acid,
hereinafter referred to as pDJA1, has been identified as a
cardiac-specific pDna1 co-chaperone and comprises the sequence of
SEQ ID NO: 1, and recombinant DNA molecules, cloned genes,
degenerate variants, mutants, analogs, or fragments thereof. This
transcript is characterized by a remarkable tissue distribution and
by a strong upregulation during ischemia/reperfusion. A yet further
embodiment of the invention provides for an isolated polypeptide,
comprising an amino acid sequence of a pDJA1 protein. The
polypeptide comprises the amino acid sequence of SEQ ID NO: 2, and
fragments, mutants, variants, analogs or derivatives thereof.
[0021] In a further embodiment of the invention, the nucleic acid
sequence of the invention may be operatively linked to an
expression control sequence and may be introduced into an
appropriate host or host cell. The invention accordingly extends to
unicellular hosts transformed with the cloned gene or recombinant
DNA molecule comprising a DNA sequence encoding the present
invention, and more particularly, the DNA sequences or fragments
thereof determined from the sequences set forth above. In a further
embodiment, the nucleic acid sequence of SEQ ID NO:1, or other
genes encoding a protein with similar activity, including
cytoprotective capacity, may be introduced into a host cell,
including but not limited to cardiac cells or neuronal stem cells,
and said cells are transplanted to the site of injury in an animal
in need of such therapy. In a preferred embodiment, the animal is a
human subject.
[0022] A twelfth aspect of the invention provides for transgenic
non-human animals (e.g., mice, rats, goats, sheep, pigs) that
express the genes and gene products of the present invention which
have the preferred activity. A preferred embodiment is a transgenic
animal having pDJA1 nucleic acids and proteins encoded by a
transgene. Transgenic, non-human knockout animals (e.g., mice), and
a pDJA1 gene and variants thereof are also provided.
[0023] A thirteenth aspect of the invention is the use of the genes
and gene products of the present invention, or use of agents that
upregulate expression and/or activity of these genes and gene
products for treatment of central nervous system (CNS) disorders,
including but not limited to stroke, Alzheimer's disease, acute and
chronic spinal cord injuries, traumatic brain injuries and other
CNS disorders.
[0024] A fourteenth aspect of the invention provides a method of
determining if a subject is at risk for developing cardiovascular
disease, said method comprising:
(I) measuring an amount of an pDJA1 gene or gene product in a
tissue sample derived from the subject, wherein said pDJA1 gene or
gene product is:
[0025] (a) a DNA corresponding to SEQ ID NO: 1, or a nucleic acid
derived therefrom;
[0026] (b) a protein comprising SEQ ID NO: 2;
[0027] (c) a nucleic acid comprising a sequence hybridizable to SEQ
ID NO: 1, or its complement under conditions of high stringency, or
a protein comprising a sequence encoded by said hybridizable
sequence;
[0028] (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1,
or its complement as determined using the NBLAST algorithm; or a
protein encoded thereby; and (II) comparing the amount of said
pDJA1 gene product in the subject with the amount of pDJA1 gene
product present in a non-ischemic cardiac tissue sample or
predetermined standard for a nonischemic cardiac tissue sample,
wherein an elevated amount of said pDJA1 gene product in the
subject compared to the amount in the non-ischemic cardiac tissue
sample or pre-determined standard for a non-ischemic cardiac tissue
sample indicates a risk of developing cardiovascular disease in the
subject.
[0029] A fifteenth aspect of the invention provides a method for
screening, diagnosis or prognosis of a cardiovascular condition
selected from the group consisting of atherosclerosis, coronary
artery disease, ischemic heart disease, myocardial infarction,
angina, stroke and other related conditions related to or resulting
from an ischemic event, said method comprising:
(I) measuring an amount of an pDJA1 gene or gene product in a
tissue sample derived from the subject, wherein said pDJA1 gene or
gene product is:
[0030] (a) a DNA corresponding to SEQ ID NO: 1, or a nucleic acid
derived therefrom;
[0031] (b) a protein comprising SEQ ID NO: 2;
[0032] (c) a nucleic acid comprising a sequence hybridizable to SEQ
ID NO: 1, or its complement under conditions of high stringency, or
a protein comprising a sequence encoded by said hybridizable
sequence;
[0033] (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1,
or its complement as determined using the NBLAST algorithm; or a
protein encoded thereby; and
(II) comparing the amount of said pDJA1 gene product in the subject
with the amount of pDJA1 gene product present in a non-ischemic
cardiac tissue sample or predetermined standard for a nonischemic
cardiac tissue sample, wherein an elevated amount of said pDJA1
gene product in the subject compared to the amount in the
non-ischemic cardiac tissue sample or pre-determined standard for a
non-ischemic cardiac tissue sample indicates a risk of developing
cardiovascular disease in the subject.
[0034] In accordance with a proposed classification of HSP40
homologues (Ohtsuka, K., et al., Mammalian HSP40/DNAJ homologs:
cloning of novel cDNAs and a proposal for their classification and
nomenclature, Cell Stress Chaperones, (2000); 5: 98-112), this
transcript has been designated pDJA1, for pig DJA1-like protein A1.
Upregulation of pDJA1 during reperfusion further expands the
concept of a program for cell survival that prevents irreversible
damage in post-ischemic myocardium.
[0035] Other objects and advantages will become apparent from a
review of the ensuing detailed description and attendant claims.
All references cited in the present application are incorporated
herein in their entirety.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIGS. 1A, 1B, 1C. Nucleotide and amino acid sequence of
pDJA1. The figure shows the full-length transcript encoding pDJA1.
The N-terminal J domain and the C-terminal prenylation site are
boxed. The four zinc fingers motifs are underlined with a solid
line. The glycine-phenylalanine stretch is underlined with a dotted
line. The AU-rich motifs in the 3'UTR are shadowed. The poly-A
signal is underlined with an arrow. The nucleic acid sequence of
pDJA1 is designated as SEQ ID NO: 1. The corresponding protein
sequence of pDJA1 is designated as SEQ ID NO: 2.
[0037] FIG. 2. Protein sequence alignment between the prototypic
human HSP40 (acc. BC002352) and pDJA1. The alignment shows that
both molecules are highly similar in the N-terminal part, which
includes the J domain and the G/F tract. PDJA1 markedly diverges
from, and is longer than, HSP40 in its C-terminal part. The protein
sequence of HSP40 is designated as SEQ ID NO: 3. The corresponding
sequence alignment of pDJA1 is designated as SEQ ID NO: 4.
[0038] FIG. 3. Tissue distribution of pDJA1 in the swine. Northern
blot performed on different pig tissues with a probe corresponding
to a 0.8-KB fragment of pDJA1 found in the subtractive
hybridization. Ribosomal RNAs (28S and 18S) are shown for equal
loading.
[0039] FIG. 4. Characterization by quantitative PCR of the
expression of pDJA1 compared to other heat-shock proteins in the
normal swine heart. Panel A shows the measurement of the pDJA1
transcript by quantitative PCR in the different cardiac chambers
(n=4 per group). **, P<0.01 versus both ventricles; *, P<0.05
versus left ventricle. Panel B shows the different expression of
pDJA1 between sub-endocardium (sub-endo) and sub-epicardium
(sub-epi) in left ventricle (n=4 per group). *, P<0.05 versus
sub-endocardium. Panel C shows that the expression of other
heat-shock proteins, such as HSP70 and HSP40, does not differ
transmurally in normal left ventricle (n=4 per group). Cyclophilin
mRNA was used as a normalizer.
[0040] FIG. 5. Upregulation of pDJA1 gene expression by
ischemia/reperfusion. Panel A shows the expression of pDJA1
measured by Northern blot in left ventricular samples from four
hearts submitted to regional ischemia for 90 min, followed by 1
hour reperfusion. In each case, a sample from the remote area and
from the ischemic area were measured in parallel. Normalization of
the pDJA1 signal to the 28S rRNA signal showed that pDJA1 was
increased 4-fold in stunned myocardium compared to remote area. *,
P<0.05 versus remote. Panel B shows the quantitative measurement
of the pDJA1 transcript in samples of the remote (open symbols) and
stunned area (closed symbols), from hearts submitted to 90 min
occlusion followed by no reperfusion, 1 h or 12 h reperfusion (n=5
in each group). The subendocardial (circles) and subepicardial
areas (squares) were measured separately. *, P<0.05 versus
corresponding value in remote myocardium. Panel C, in-situ
hybridization (magnification, .times.40) showing that the
expression of the pDJA1 gene in stunned myocardium is
myocyte-specific.
[0041] FIG. 6. Cytoprotective effect of pDJA1 in isolated cardiac
myocytes. Isolated cardiac myocytes were infected with an
adenovirus containing the coding sequence of pDJA1 (Ade-pDJA1), and
compared with an adenovirus harboring an irrelevant sequence
(Ade-.beta.Gal vector). Apoptosis was induced by addition of 4
.mu.M staurosporine for 1 hour and quantified by the measurement of
caspase-3 activation (n=3). Values are expressed as the increase in
apoptotic rate in both groups after addition of staurosporine
(-staur. vs +staur.)*, P<0.05 versus corresponding value in
Ade-.beta.Gal group.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Before the present methods and treatment methodology are
described, it is to be understood that this invention is not
limited to particular methods, and experimental conditions
described, as such methods and conditions may vary. It is also to
be understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only in the appended claims.
[0043] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described. All publications
mentioned herein are incorporated herein by reference.
DEFINITIONS
[0045] The terms used herein have the meanings recognized and known
to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth
below.
[0046] "Agent" refers to all materials that may be used to prepare
pharmaceutical and diagnostic compositions, or that may be
compounds, nucleic acids, polypeptides, fragments, isoforms,
variants, or other materials that may be used independently for
such purposes, all in accordance with the present invention.
[0047] The term "antibody" as used herein includes intact molecules
as well as fragments thereof, such as Fab and F(ab').sub.2, which
are capable of binding the epitopic determinant. Antibodies that
bind the genes or gene products of the present invention can be
prepared using intact polynucleotides or polypeptides or fragments
containing small peptides of interest as the immunizing antigen
attached to a carrier molecule. Commonly used carriers that are
chemically coupled to peptides include bovine serum albumin and
thyroglobulin. The coupled peptide is then used to immunize the
animal (e.g, a mouse, rat or rabbit). The antibody may be a
"chimeric antibody", which refers to a molecule in which different
portions are derived from different animal species, such as those
having a human immunoglobulin constant region and a variable region
derived from a murine mAb. (See, e.g., Cabilly et al., U.S. Pat.
No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397.). The
antibody may be a human or a humanized antibody. The antibody may
be prepared in mice, rats, goats, sheep, swine, dogs, cats, or
horses.
[0048] A "therapeutically effective amount" is an amount sufficient
to decrease or prevent the symptoms associated with the cardiac or
other related conditions contemplated for therapy with the
compositions of the present invention.
[0049] A "variant" (v) of polynucleotides or polypeptides, as the
term is used herein, are polynucleotides or polypeptides that are
different from a reference polynucleotide or polypeptide,
respectively. Variant polynucleotides are generally limited so that
the nucleotide sequence of the reference and the variant are
closely related overall and, in many regions, identical. Changes in
the nucleotide sequence of the variant may be silent. That is, they
may not alter the amino acid sequence encoded by the
polynucleotide. Where alterations are limited to silent changes of
this type a variant will encode a polypeptide with the same amino
acid sequence as the reference. Alternatively, changes in the
nucleotide sequence of the variant may alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Such nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions, and truncations in the polypeptide
encoded by the reference sequence. Variant polypeptides are
generally limited so that the sequences of the reference and the
variant are that are closely similar overall and, in many regions,
identical. For example, a variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, deletions, fusions, and truncations, which may be
present or absent in any combination. Such variants can differ in
their amino acid composition (e.g. as a result of allelic or
natural variation in the amino acid sequence, e.g. as a result of
alternative mRNA or pre-mRNA processing, e.g. alternative splicing
or limited proteolysis) and in addition, or in the alternative, may
arise from differential post-translational modification (e.g.,
glycosylation, acylation, phosphorylation, isoprenylation,
lipidation).
[0050] "Gene Product" as used herein, unless otherwise indicated,
is a protein or polypeptide encoded by the nucleic acid sequences
identified by the methods of the present invention, including but
not limited to SEQ ID NO: 2; a nucleic acid comprising a sequence
hybridizable to SEQ ID NO: 1 or its complement under conditions of
high stringency, or a protein comprising a sequence encoded by said
hybridizable sequence; a nucleic acid at least 90% homologous to
SEQ ID NO: 1 or its complement as determined using the NBLAST
algorithm; a nucleic acid at least 90% homologous to SEQ ID NO: 1
or a fragment or derivative of any of the foregoing proteins or
nucleic acids.
[0051] "Modulate" as used herein, refers to a compound or agent
(including but not limited to proteins, polypeptides, or fragments
thereof, nucleotides, nucleic acid fragments, synthetic organic
compounds, antibodies) which are capable of increasing or
decreasing the level and/or activity of a gene or gene product
identified by the methods described herein, said genes or gene
products having a beneficial effect in preventing cell death and/or
irreversible damage in cardiovascular disease, or other diseases or
conditions whereby ischemia results in damage to tissue, including
heart tissue, brain tissue or other tissues affected by a lack of
oxygen due to inadequate perfusion. These may include
atherosclerosis or restenotic lesions, stroke, or anemia, to name a
few non-limiting examples. Those skilled in the art, based on the
present description, will understand that such modulation can be
determined by assays and techniques known to those of skill in the
art, including as described in more detail herein.
[0052] "Agonist" as used herein, refers to a compound or agent
(including but not limited to proteins, polypeptides, or fragments
thereof, nucleotides, nucleic acid fragments, synthetic organic
compounds, antibodies) capable of increasing the level and/or
activity of a pDJA1-like molecule or a variant thereof and may be
referred to herein as an agonist.
[0053] "Analog" as used herein, refers to a nucleotide, a protein,
or a polypeptide that possesses similar or identical activity or
function(s) as the nucleotide, protein or polypeptide having the
desired activity and therapeutic effect of the present invention
(eg. protection of cells from death and/or prevention of
irreversible damage in post-ischemic events in tissues), but need
not necessarily comprise a sequence that is similar or identical to
the sequence of the preferred embodiment, such as that of SEQ ID
NOS: 1 and 2, or possess a structure that is similar or identical
to that of SEQ ID NOS: 1 and 2. As used herein, a nucleic acid or
nucleotide sequence, or an amino acid sequence of a protein or
polypeptide is "similar" to that of a nucleic acid, nucleotide or
protein or polypeptide having the desired activity if it satisfies
at least one of the following criteria: (a) the nucleic acid,
nucleotide, protein or polypeptide has a sequence that is at least
30% (more preferably, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99%) identical to the nucleic acid, nucleotide,
protein or polypeptide sequences having the desired activity as
described herein (b) the polypeptide is encoded by a nucleotide
sequence that hybridizes under stringent conditions to a nucleotide
sequence encoding at least 5 amino acid residues (more preferably,
at least 10 amino acid residues, at least 15 amino acid residues,
at least 20 amino acid residues, at least 25 amino acid residues,
at least 40 amino acid residues, at least 50 amino acid residues,
at least 60 amino residues, at least 70 amino acid residues, at
least 80 amino acid residues, at least 90 amino acid residues, at
least 100 amino acid residues, at least 125 amino acid residues, or
at least 150 amino acid residues) of the AAPI; or (c) the
polypeptide is encoded by a nucleotide sequence that is at least
30% (more preferably, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99%) identical to the nucleotide sequence encoding
the polypeptides of the present invention having the desired
therapeutic effect. As used herein, a polypeptide with "similar
structure" to that of the preferred embodiments of the invention
refers to a polypeptide that has a similar secondary, tertiary or
quarternary structure as that of the preferred embodiment (eg. SEQ
ID NO: 2). The structure of a polypeptide can determined by methods
known to those skilled in the art, including but not limited to,
X-ray crystallography, nuclear magnetic resonance, and
crystallographic electron microscopy.
[0054] "Derivative" refers to either a protein or polypeptide that
comprises an amino acid sequence of a parent protein or polypeptide
that has been altered by the introduction of amino acid residue
substitutions, deletions or additions, or a nucleic acid or
nucleotide that has been modified by either introduction of
nucleotide substitutions or deletions, additions or mutations. The
derivative nucleic acid, nucleotide, protein or polypeptide
possesses a similar or identical function as the parent
polypeptide.
[0055] "Fragment" refers to either a protein or polypeptide
comprising an amino acid sequence of at least 5 amino acid residues
(preferably, at least 10 amino acid residues, at least 15 amino
acid residues, at least 20 amino acid residues, at least 25 amino
acid residues, at least 40 amino acid residues, at least 50 amino
acid residues, at least 60 amino residues, at least 70 amino acid
residues, at least 80 amino acid residues, at least 90 amino acid
residues, at least 100 amino acid residues, at least 125 amino acid
residues, at least 150 amino acid residues, at least 175 amino acid
residues, at least 200 amino acid residues, or at least 250 amino
acid residues) of the amino acid sequence of a parent protein or
polypeptide, or a nucleic acid comprising a nucleotide sequence of
at least 10 base pairs (preferably at least 20 base pairs, at least
30 base pairs, at least 40 base pairs, at least 50 base pairs, at
least 50 base pairs, at least 100 base pairs, at least 200 base
pairs) of the nucleotide sequence of the parent nucleic acid. Any
given fragment may or may not possess a functional activity of the
parent nucleic acid or protein or polypeptide.
[0056] The "percent identity" of two amino acid sequences or of two
nucleic acid sequences can be or is generally determined by
aligning the sequences for optimal comparison purposes (e.g., gaps
can be introduced in either sequences for best alignment with the
other sequence) and comparing the amino acid residues or
nucleotides at corresponding positions. The "best alignment" is an
alignment of two sequences that results in the highest percent
identity. The percent identity is determined by the number of
identical amino acid residues or nucleotides in the sequences being
compared (i.e., % identity=# of identical positions/total # of
positions.times.100).
[0057] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm known to those
of skill in the art. An example of a mathematical algorithm for
comparing two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.
Biol. 215:403-410 have incorporated such an algorithm. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov.
[0058] Another example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
CABIOS (1989). The ALIGN program (version 2.0) which is part of the
GCG sequence alignment software package has incorporated such an
algorithm. Other algorithms for sequence analysis known in the art
include ADVANCE and ADAM as described in Torellis and Robotti
(1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within
FASTA, ktup is a control option that sets the sensitivity and speed
of the search.
[0059] "Diagnosis" refers to diagnosis, prognosis, monitoring,
characterizing, selecting patients, including participants in
clinical trials, and identifying patients at risk for or having a
particular disorder or clinical event or those most likely to
respond to a particular therapeutic treatment, or for assessing or
monitoring a patient's response to a particular therapeutic
treatment.
[0060] "Treatment" refers to therapy, prevention and prophylaxis
and particularly refers to the administration of medicine or the
performance of medical procedures with respect to a patient, for
either prophylaxis (prevention) or to cure or reduce the extent of
or likelihood of occurrence of the infirmity or malady or condition
or event in the instance where the patient is afflicted.
[0061] In accordance with a proposed classification of HSP40
homologues (Ohtsuka, K., et al., Mammalian HSP40/DNAJ homologs:
cloning of novel cDNAs and a proposal for their classification and
nomenclature, Cell Stress Chaperones, (2000); 5: 98-112), the
transcript of the present invention has been designated pDJA1, for
pig DJA1-like protein A1.
General Description
[0062] The present invention relates to the discovery that a gene,
pDJA1 and variants thereof, have an expression pattern that is
up-regulated in cardiac tissue following a period of ischemia
followed by reperfusion, and in cardiac cell lines. The invention
relates to the use of said gene, gene products, and agonists of
said gene or gene products (pDJA1 and variants thereof, cDNA, RNA,
and/or protein, small synthetic organic molecules, antibodies) as
targets for diagnosis, drug screening and therapies for
cardiovascular diseases. In a preferred embodiment, the invention
provides for methods of using the protein and variants thereof, or
nucleic acids that encode said proteins for the treatment,
prevention and diagnosis of cardiovascular disease.
[0063] In particular, the methods of the present invention include
using nucleic acid molecules that encode the pDJA1 protein and
variants thereof, and recombinant DNA molecules, cloned genes or
degenerate variants thereof, and in particular naturally occurring
variants that encode pDJA1 related gene products. The methods of
the present invention additionally include using cloning vectors,
including expression vectors, containing the nucleic acid molecules
encoding pDJA1 and variants thereof, and hosts that contain such
nucleic acid molecules. The methods of the present invention also
encompass the use of pDJA1 gene products and variants thereof,
including fusion proteins, and antibodies directed against such
pDJA1 gene products or conserved variants or fragments thereof.
[0064] This novel gene, designated "pDJA1", which has been cloned,
was identified using subtractive hybridization between stunned and
normal heart tissue in a pig model for ischemia/reperfusion. This
gene is expressed in heart tissue which was transiently deprived of
oxygen (ischemia) followed by reperfusion. The 0.8-kb fragment,
which was subcloned did not match any known transcript in public
databases. Further studies that were done to determine the
full-length sequence of the novel gene, identified the cDNA as
being 3.1-kb long and characterized by a 62 nucleotide long 5'-UTR,
a 397 amino acid open reading frame and a 1.75 kb long 3'-UTR.
Furthermore, the open reading frame begins with an ATG nucleotide
at nucleotide 63, and is not preceded by a Kozak's consensus for
translation initiation. The protein encoded by this nucleic acid
has an apparent molecular weight of 44.7 kDa and a pI=8.27. The
nucleic acid sequence of this novel gene is found in SEQ ID NO: 1.
The corresponding amino acid sequence encoded by this gene is found
in SEQ ID NO: 2.
[0065] pDJA1 as described herein, is a novel heart-specific
ventricle-enriched cardioprotective co-chaperone, which
participates in the program of cell survival that limits
irreversible damage in post-ischemic myocardium. The findings in
the present application suggest that this gene, its gene product,
and other agents or agonists that have the same activity and/or
function in a similar manner may prove to be useful in the
treatment of cardiovascular disease, ischemic heart disease,
myocardial infarct or related disorders.
[0066] Furthermore, the genes, gene products, or other agents or
agonists may also prove useful in a diagnostic setting in order to
monitor patients believed to have experienced a myocardial infarct
or ischemic cardiac event or other related cardiac condition, and
may be used in a prognostic manner to determine the potential for
subsequent ischemic cardiac events. A search for agonists of this
gene may prove to be a useful strategy for identifying a new class
of cardioprotective agents and treatment modalities.
[0067] Thus, the present invention further relates to methods for
the diagnostic evaluation and prognosis of cardiovascular disease
in a subject animal. Preferably the subject is a mammal, more
preferably the subject is a human. In a preferred embodiment the
invention relates to methods for diagnostic evaluation and
prognosis of cardiovascular disease. For example, nucleic acid
molecules of the invention can be used as diagnostic hybridization
probes or as primers for diagnostic PCR analysis for detection of
abnormal expression of the pDJA1 gene.
[0068] Antibodies or other binding partners to pDJA1 and variants
thereof can be used in a diagnostic test to detect the presence of
the pDJA1 gene or gene product in body fluids, cells or in tissue
biopsy. In specific embodiments, measurement of serum or cellular
pDJA1 gene products and variants thereof can be made to detect
cellular and/or tissue damage following a myocardial infarct, a
stroke, or other related cardiovascular diseases or conditions.
[0069] The present invention also relates to methods for the
identification of subjects having a predisposition to
cardiovascular disease, or alternatively, being at risk for a
second myocardial infarct or stroke or related condition. The
subject can be any animal, but preferably the subject is a mammal,
and most preferably the subject is a human. In a non-limiting
example nucleic acid molecules of the invention can be used as
diagnostic hybridization probes or as primers for quantitative
reverse transcriptase-PCR (RT-PCR) analysis to determine expression
levels of the pDJA1 gene or gene product and variants thereof. In
another example, nucleic acid molecules of the invention can be
used as diagnostic hybridization probes or as primers for
diagnostic PCR analysis for the identification of pDJA1 and
variants thereof, naturally occurring or non-naturally occurring
gene mutations, allelic variations and regulatory defects in the
pDJA1 gene.
[0070] In a preferred embodiment, the present invention further
provides methods of determining if a subject is at risk for
developing cardiovascular disease, said method comprising (I)
measuring an amount of an pDJA1 gene product in a sample derived
from the subject, wherein said pDJA1 gene product is: (a) an DNA
corresponding to SEQ ID NO: 1, or a nucleic acid derived therefrom;
(b) a protein comprising SEQ ID NO: 2; (c) a nucleic acid
comprising a sequence hybridizable to SEQ ID NO: 1, or its
complement under conditions of high stringency, or a protein
comprising a sequence encoded by said hybridizable sequence; (d) a
nucleic acid at least 90% homologous to SEQ ID NO: 1, or its
complement as determined using the NBLAST algorithm; or a protein
encoded thereby; and (II) comparing the amount of said pDJA1 gene
product in the subject with the amount of pDJA1 gene product
present in a non-ischemic cardiac tissue sample or predetermined
standard for a nonischemic cardiac tissue sample, wherein an
elevated amount of said pDJA1 gene product in the subject compared
to the amount in the non-ischemic cardiac tissue sample or
pre-determined standard for a non-ischemic cardiac tissue sample
indicates a risk of developing cardiovascular disease in the
subject.
[0071] Imaging methods, for imaging the localization and/or amounts
of pDJA1 gene products in a patient, are also provided for
diagnostic and prognostic use.
Screening Assays
[0072] Intensive and systematic evaluation of gene expression
patterns is essential in understanding the physiological mechanisms
associated with cell death and/or cellular responsiveness to the
events leading to cell death or tissue damage following an ischemic
cardiac episode. Several techniques that permit comparison of gene
expression in normal and damaged cells are known in the art.
Examples of these techniques include: Serial Analysis of Gene
Expression (SAGE) (Velculescu et al., 1995, Science 270:484);
Restriction Enzyme Analysis of Differentially Expressed Sequences
(READS) (Prasher et al., 1999, Methods in Enzymology 303:258);
Amplified Fragment Length Polymorphism (AFLP) (Bachem et al., 1996,
Plant Journal 9:745); Representational Difference Analysis (RDA)
(Hubank et al., 1994, Nucleic Acid Research 22:(25):5640);
differential display (Liang et al., 1992, Cancer Research
52(24):6966); and suppression subtractive hybridization (SSH)
(Diatchenko et al., 1996, Proc. Natl. Acad. Sci. USA 93:6025). The
use of such differential expression methods have led the present
inventors to the identification and characterization of the pDJA1
gene, as a gene whose expression is associated with heart tissue
damaged by a transient period of ischemia followed by reperfusion.
This discovery by the present inventors has made possible the use
of pDJA1 and variants thereof for the treatment, prevention and
diagnosis of cardiovascular disease, including but not limited to
atherosclerosis, coronary artery disease, ischemic heart disease,
myocardial infarct, stroke and other related conditions.
Hybridization Conditions
[0073] A nucleic acid which is hybridizable to an PDJA1 nucleic
acid (e.g., having a sequence as set forth in SEQ ID NO: 1, or to
its reverse complement, or to a nucleic acid encoding an PDJA1
derivative, or to its reverse complement under conditions of low
stringency can be used in the methods of the invention to detect
the presence of an PDJA1 gene and/or presence or expression level
of an PDJA1 gene product. By way of example and not limitation,
procedures using such conditions of low stringency are as follows
(see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A.
78, 6789-6792). Filters containing DNA are pretreated for 6 h at
40.degree. C. in a solution containing 35% formamide, 5.times.SSC,
50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA,
and 500 .mu.g/ml denatured salmon sperm DNA. Hybridizations are
carried out in the same solution with the following modifications:
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA,
10% (wt/vol) dextran sulfate, and 5-20.times.10.sup.6 cpm
.sup.32P-labeled probe is used. Filters are incubated in
hybridization mixture for 18-20 h at 40.degree. C., and then washed
for 1.5 h at 55.degree. C. in a solution containing 2.times.SSC, 25
mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is
replaced with fresh solution and incubated an additional 1.5 h at
60.degree. C. Filters are blotted dry and exposed for
autoradiography. If necessary, filters are washed for a third time
at 65-68.degree. C. and re-exposed to film. Other conditions of low
stringency that may be used are well known in the art (e.g., as
employed for cross-species hybridizations).
[0074] A nucleic acid which is hybridizable to an PDJA1 nucleic
acid (e.g., having a sequence as set forth in SEQ ID NO: 1 or to
its reverse complement, or to a nucleic acid encoding an PDJA1
derivative, or to its reverse complement under conditions of high
stringency) is also provided for use in the methods of the
invention. By way of example and not limitation, procedures using
such conditions of high stringency are as follows. Prehybridization
of filters containing DNA is carried out for 8 h to overnight at
65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48
h at 65.degree. C. in prehybridization mixture containing 100
.mu.g/ml denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of
.sup.32P-labeled probe. Washing of filters is done at 37.degree. C.
for 1 h in a solution containing 2.times.SSC, 0.01% PVP, 0.01%
Ficoll, and 0.01% BSA. This is followed by a wash in 0.1.times.SSC
at 50.degree. C. for 45 min before autoradiography. Other
conditions of high stringency that may be used are well known in
the art.
[0075] A nucleic acid which is hybridizable to an PDJA1 nucleic
acid (e.g., having a sequence as set forth in SEQ ID NO: 1 or to
its reverse complement, or to a nucleic acid encoding an PDJA1
derivative, or to its reverse complement under conditions of
moderate stringency) is also provided for use in the methods of the
invention. For example, but not limited to, procedures using such
conditions of moderate stringency are as follows: filters
comprising immobilized DNA are pretreated for 6 hours at 55.degree.
C. in a solution containing 6.times.SSC, 5.times.Denhardt's
solution, 0.5% SDS and 100 .mu.g/ml denatured salmon sperm DNA.
Hybridizations are carried out in the same solution with
5-20.times.10.sup.6 cpm .sup.32P-labeled probe. Filters are
incubated in hybridization mixture for 18-20 hours at 55.degree.
C., and then washed twice for 30 minutes at 60.degree. C. in a
solution containing 1.times.SSC and 0.1% SDS. Filters are blotted
dry and exposed for autoradiography. Washing of filters is done at
37.degree. C. for 1 hour in a solution containing 2.times.SSC, 0.1%
SDS. Other conditions of moderate stringency that may be used are
well known in the art. (see, e.g., Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al., eds., in
the Current Protocols in Molecular Biology series of laboratory
technique manuals, 1987-1997 Current Protocols.COPYRGT. 1994-1997
John Wiley and Sons, Inc.).
[0076] The invention provides methods for identifying agents (e.g.,
chemical compounds, carbohydrates, proteins, peptides, antibodies
or nucleotides) that enhance the expression and/or activity of
pDJA1 gene or gene products. The invention also provides methods of
identifying agents, candidate compounds or test compounds that
specifically bind to pDJA1. Examples of agents, candidate compounds
or test compounds include, but are not limited to, nucleic acids
(e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides,
peptidomimetics, small molecules and other drugs. Agents can be
obtained using any of the numerous suitable approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
1997, Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and
U.S. Pat. No. 5,807,683, each of which is incorporated herein in
its entirety by reference).
[0077] In one embodiment, agents that interact with (i.e., bind to)
pDJA1 or a polypeptide or fragment (e.g. a functionally active
fragment), are identified in a cell-based assay system. In
accordance with this embodiment, cells expressing pDJA1 comprising
an PDJA1 peptide or polypeptide, a fragment thereof, are contacted
with a candidate compound or a control compound and the ability of
the candidate compound to interact with pDJA1 is determined. If
desired, this assay may be used to screen a plurality (e.g., a
library) of candidate compounds. The cell, for example, can be of
prokaryotic or eukaryotic origin (e.g., E. coli or CHO cells), and
may contain the pDJA1 peptide or polypeptide, fragment, or related
polypeptide thereof. In some embodiments, the pDJA1 gene or pDJA1
polypeptide, fragment, or related polypeptide thereof or the
candidate compound is labeled, for example with a radioactive label
(such as .sup.32P, .sup.35S or .sup.125I) or a fluorescent label
(such as fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to
enable detection of an interaction between a pDJA1 and a candidate
compound. The ability of the candidate compound to interact
directly or indirectly with the pDJA1 can be determined by methods
known to those of skill in the art. For example, the interaction
can be determined by flow cytometry, a scintillation assay,
immunoprecipitation or western blot analysis.
[0078] In another embodiment, agents interact with (i.e., bind to)
the pDJA1 gene or gene product in a cell-free assay system. In
accordance with this embodiment, pDJA1 is contacted with a
candidate compound or a control compound and the ability of the
candidate compound to interact with the pDJA1 is determined. If
desired, this assay may be used to screen a plurality (e.g. a
library) of candidate compounds. In one embodiment, the pDJA1 gene
or gene product is first immobilized, by, for example, contacting
the pDJA1 with an immobilized antibody which specifically
recognizes and binds it, or by contacting a purified preparation of
the pDJA1 with a surface designed to bind nucleic acids or
proteins. The pDJA1 may be partially or completely purified (e.g.,
partially or completely free of other nucleic acids or
polypeptides) or part of a cell lysate. The ability of the
candidate compound to interact with the pDJA1 can be determined by
methods known to those of skill in the art.
[0079] In another embodiment, a cell-based assay system is used to
identify agents that bind to or modulate the expression or activity
of the pDJA1 gene or gene product, or a biologically active portion
thereof. In a primary screen, a plurality (e.g., a library) of
compounds are contacted with cells that naturally express pDJA1 in
order to identify compounds that modulate the expression and/or
activity of the pDJA1. The ability of the candidate compound to
modulate the expression and/or activity of the pDJA1 can be
determined by methods known to those of skill in the art, including
without limitation, flow cytometry, a scintillation assay,
immunoprecipitation and western blot analysis.
[0080] In another embodiment, agents that modulate (i.e.,
up-regulate or down-regulate) the expression and/or activity of
PDJA1 are identified by contacting cells (e.g., cells of
prokaryotic or eukaryotic origin) containing the components capable
of forming an active pDJA1 with a candidate compound or a control
compound (e.g., phosphate buffered saline (PBS)) and determining
the expression and/or activity of the pDJA1. The level of pDJA1
expression and/or PDJA1 activity in the presence of the candidate
compound is compared to the level of expression or activity in the
absence of the candidate compound (e.g., in the presence of a
control compound). The candidate compound can then be identified as
a modulator of the expression and/or assembly of the pDJA1 based on
this comparison. For example, when presence of an active pDJA1 is
significantly greater in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of pDJA1 expression/formation and/or an enhancer of
pDJA1 activity. Alternatively, when presence of an active pDJA1 is
significantly less in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of pDJA1 expression/formation and/or inhibitor of pDJA1
activity.
[0081] In another embodiment, agents that modulate (i.e.,
up-regulate or down-regulate) the expression, activity or both, of
pDJA1 are identified in an animal model. Examples of suitable
animals include, but are not limited to, mice, rats, pigs, rabbits,
monkeys, guinea pigs, dogs and cats. Preferably, the animal used
represents a model of a pDJA1-associated cardiovascular disease,
such as ischemic heart disease, stroke, myocardial infarct.
[0082] In accordance with this embodiment, the test compound or a
control compound is administered (e.g. orally, intravenously,
intramuscularly, subcutaneously, intrathecally, rectally) to a
suitable animal and the effect on the expression or activity or
both expression and activity of the pDJA1 is determined, or the
effect on an pDJA1-bearing target cell is determined. Changes in
the expression and/or activity of pDJA1 can be assessed by any
suitable method described above, based on the present
description.
[0083] This invention further provides novel agents identified by
the above-described screening assays and uses thereof for
treatments as described herein.
Therapeutic Uses of the Invention
[0084] Another aspect of the invention provides for the use of
pDJA1 genes or gene products and proteins or polypeptides, or
agonists thereof in prevention of cell death in vitro and in vivo.
One embodiment of the invention features use of the genes and/or
gene products, protein, polypeptides, small molecule agonists to
prevent or reverse tissue damage in the heart of a subject having
or prone to having cardiovascular disease. The agonists of pDJA1
expression and/or activity are envisioned to be small molecule
inhibitors, peptides, polypeptides, antibodies, antibody fragments
or mimics thereof.
[0085] The invention provides for treatment or prevention of
various cardiac diseases and disorders by administration of a
therapeutic agent. Such agents include but are not limited to:
agents which enhance expression, formation or activity of pDJA1,
agents which modulate the activity of pDJA1, agents able to act as
agonists of pDJA1, and related analogs, derivatives, and fragments
thereof. Such agonists may include small molecule agonists.
[0086] In one embodiment wherein expression and/or activity of
pDJA1 is desirable, one or more agents that upregulate pDJA1 gene
expression and/or gene or gene product activity, are administered
alone or in combination with one or more additional therapeutic
compounds or treatments. In a preferred embodiment, an upregulator
of PDJA1 gene or gene product activity is administered to a human
subject for therapy (e.g. to ameliorate symptoms or to retard onset
or progression) of cardiovascular disease.
Assays for Therapeutic Compounds
[0087] The present invention also provides for assays for use in
discovery of pharmaceutical products in order to identify or verify
the efficacy of compounds for treatment or prevention of
cardiovascular diseases in which pDJA1 may prove efficacious. In
one embodiment, agents can be assayed for their ability to inhibit
cell death in vitro or in vivo. Compounds able to enhance
expression or activity of pPDJA1 in vitro can be further tested for
in vivo activity in experimental animal models of cardiovascular
disease and can be used as lead compounds for further drug
discovery, or used therapeutically.
[0088] In various embodiments, in vitro assays can be carried out
with cardiac cells, containing the pDJA1 gene and which are
representative of the cell type involved in a subject's disease, to
determine if a compound has a desired effect upon such cell types.
In one embodiment, the cells are derived from cardiac tissue, such
as myocytes.
[0089] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to rats, mice, pigs, chicken, cows, monkeys, rabbits, etc.
For in vivo testing, prior to administration to humans, any animal
model system known in the art may be used. In one embodiment, test
compounds that modulate the expression or activity of pDJA1 are
identified in non-human animals (e.g., mice, rats, pigs, monkeys,
rabbits, and guinea pigs), preferably non-human animal models for
cardiovascular diseases. In accordance with this embodiment, a test
compound or a control compound is administered to the animals, and
the effect of the test compound on pDJA1 levels or activity is
determined in cells obtained from the animal. A test compound that
alters the level or activity of pDJA1 can be identified by
comparing the level of the selected pDJA1 in a cell culture
obtained from an animal or group of animals treated with a test
compound with the level of the pDJA1 in a cell culture obtained
from an animal or group of animals treated with a control
compound.
[0090] In yet another embodiment, test compounds that modulate the
level or activity of pDJA1 are identified in human subjects having
a cardiovascular disease or condition associated with expression of
pDJA1. In accordance with this embodiment, a test compound or a
control compound is administered to the human subject, and the
effect of a test compound on either reduction in damage to heart
tissue, or amelioration of symptoms associated with the disease is
determined by methods known in the art.
Therapeutic and Prophylactic Compositions and Their Use
[0091] The invention provides methods of treatment comprising
administering to a subject an effective amount of an agent of the
invention. In a preferred aspect, the compound is substantially
purified (e.g., substantially free from substances that limit its
effect or produce undesired side-effects). The subject is
preferably an animal, including but not limited to animals such as
monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is
preferably a mammal, and most preferably human. In one specific
embodiment, a non-human mammal is the subject. In another specific
embodiment, a human mammal is the subject.
[0092] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, or microcapsules. Methods of
introduction can be enteral or parenteral and include but are not
limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, topical and oral
routes. The compounds may be administered by any convenient route,
for example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent. In a
specific embodiment, it may be desirable to administer the
pharmaceutical compositions of the invention locally to the area in
need of treatment.
[0093] Another aspect of the invention provides for pharmaceutical
compositions comprising purified pDJA1 enhancers for therapeutic
use in treatment of cardiovascular diseases. One embodiment
features treatment of a wide range of cardiovascular diseases or
conditions with pharmaceutical compositions containing acceptable
carriers and excipients.
[0094] Such compositions comprise a therapeutically effective
amount of an agent, and a pharmaceutically acceptable carrier. In a
particular embodiment, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the therapeutic is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like. The composition can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. Such
compositions will contain a therapeutically effective amount of the
compound, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the subject. The formulation should suit the mode
of administration.
[0095] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0096] The amount of the compound of the invention which will be
effective in the treatment of cardiovascular diseases can be
determined by standard clinical techniques based on the present
description. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each subject's circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20-500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0097] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects (a) approval by the agency of manufacture,
use or sale for human administration, (b) directions for use, or
both.
[0098] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved, for example, and
not by way of limitation, by local infusion during surgery, by
topical application, by injection, by means of a catheter, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers or co-polymers such as Elvax (see Ruan et al,
1992, Proc Natl Acad Sci USA, 89:10872-10876). In one embodiment,
administration can be by direct injection by aerosol inhaler.
[0099] In another embodiment, the compound can be delivered in a
vesicle, in particular a liposome (see Langer (1990) Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0100] In yet another embodiment, the compound can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989)
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J. (1983) Macromol. Sci. Rev. Macromol. Chem. 23:61;
see also Levy et al. (1985) Science 228:190; During et al. (1989)
Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In
yet another embodiment, a controlled release system can be placed
in proximity of the therapeutic target, i.e., the airways, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release (1984) supra, vol. 2,
pp. 115-138). Other suitable controlled release systems are
discussed in the review by Langer (1990) Science 249:1527-1533.
EXAMPLES
Animal Model for Ischemia/Reperfusion
[0101] Female domestic swine (22-25 kg) were anesthetized with
thiopental sodium (5-10 mg/kg, i.v.) and isoflurane (0.5-1.5 vol
%). A left thoracotomy was performed through the fifth intercostal
space to expose the heart.sup.16. A hydraulic occluder was
implanted around the base of the left anterior descending (LAD)
artery. Myocardial blood flow through the LAD was monitored by a
Doppler flow probe. After 3 days of recovery, stunning was induced
in the conscious animal by inflating the coronary occluder, to
reduce the blood flow in the LAD by 40%. Reduction of the blood
flow was controlled on-line via the flow probe. The coronary
stenosis was maintained for 90 min, followed by deflation of the
occluder and full reperfusion. Animals were anesthetized at the end
of the 90 min-stenosis period (n=5), or after 1 h (n=5) and 12 h
(n=5) reperfusion. In each case, myocardial samples were taken from
both the stunned area (centrally in the LAD territory) and the
remote area of the beating heart. Each sample was further separated
in a subendocardial and a subepicardial portion. Three instrumented
pigs, in which no occlusion was performed, were used as shams.
Samples from both atria and from different organs (kidney, liver,
lung, spleen, aorta, skeletal muscle, stomach) were taken as well.
The samples were frozen in liquid nitrogen or fixed in fresh 4%
paraformaldehyde. The investigation conforms with the Guide for the
Care and Use of Laboratory Animals published by the US National
Institute of Health (NIH Publication No 85-23, revised 1996).
[0102] Cloning of pDJA1
[0103] RNA Extraction
[0104] About 300 mg of each sample was homogenized in 3 ml of the
guanidium thiocyanate-phenol-chloroform solution (Triazol, Gibco
Life Technologies). Total RNA was extracted (Chomczynski, P., et
al., Single-step method of RNA isolation by acid guanidium
thiocyanate-phenol-chloroform extraction, Anal Biochem., (1987);
162: 159-169), resuspended in 50 .mu.l DEPC-water, and its
concentration was measured spectrophotometrically by the absorbance
at 260 nm. The integrity of the RNA pool was checked on a
1%-agarose denaturing gel stained with ethidium bromide.
[0105] Cloning Protocol for PDJA1
[0106] A pig heart cDNA library was subcloned in the pCMV Sport6
vector (Life Technologies) and used for screening with primers
designed from the sequence obtained in the subtractive
hybridization. The cDNA was obtained by PCR cloning and colony
hybridization. The 5' end of the transcript was obtained by 5'RACE
after decapping of the transcripts (First Choice RLM-RACE, Ambion).
PCR products were sequenced by triple pass on a 3100 Genetic
Analyzer (Applied Biosystems) using the Big-Dye Terminator (Applied
Biosystems). Data analysis was performed with the ABI AutoAssembler
software. Gene analysis and sequence comparisons were performed
with the MacVector software.
[0107] Northern Blotting
[0108] Fifteen micrograms of total RNA was applied on a
1.2%-agarose denaturing gel stained with ethidium bromide. After
migration, the RNA was transferred overnight to a nylon Hybond-N
membrane (Amersham Pharmacia), then cross-linked by UV. A probe was
derived as an isolated restriction fragment from the subtractive
library, heat-denatured, and labeled with [.alpha..sup.32P]-dCTP
(Prime-It II kit, Stratagene). Hybridization was performed
overnight at 42.degree. C. in a hybridization solution containing
50% formamide. Intensity of the radioactive signal was measured
with the Multi-Analyst detection system (Biorad). RNA integrity was
controlled by comparison of the bands corresponding to the 28S and
18S rRNAs.
[0109] Quantitative RT-PCR
[0110] Expression of pDJA1 was measured by quantitative RT-PCR on a
7700 Sequence Detector (Applied Biosystems) with specific primers
(forward:5'-CTCTCTTGGAAGCTTCCTGAAC-3' (SEQ ID NO: 5),
REVERSE:5'-GCACTGCAAAGGCTGTCAA-3'(SEQ ID NO: 6)) and a fluorescent
probe (5'FAM--AAGCTTGTGGTGAGGACAAACCAGTGTTT-3'TAMRA (SEQ ID NO:
7)). The mRNA of interest was reverse-transcribed from 60 nanograms
of total RNA, and subsequently used for quantitative 2-step PCR (40
cycles of a 10 sec-step at 95.degree. C. and a 1 min-step at
60.degree. C.). Internal RNA standards were prepared from the
PCR-amplified cDNA after ligation of the T7 promoter using the
MegaShortScript kit (Ambion, Austin, Tex.) (Depre, C., et al.,
Unloaded heart in vivo replicates fetal gene expression cardiac
hypertrophy, Nature Medicine, (1998); 4: 1269-1275). The values of
the transcript were normalized to the transcript level of
cyclophilin, measured in each sample as an internal control.
[0111] In-Situ Hybridization
[0112] Samples were fixed in 4% paraformaldehyde/PBS, embedded in
paraffin and sectioned at 6-.mu.m intervals. Sections were dewaxed,
rehydrated in ethanol, and treated with 0.8% pepsin in 0.2N HCl
(DAKO) for 5 min at 37.degree. C., followed by a 5-min rinse in
H.sub.2O, Sections were then re-fixed for 20 min in 4%
paraformaldehyde dissolved in PBS. After washing, sections were
acetylated in 0.25% acetic anhydride diluted in 0.1M
triethanolamine buffer (pH=8.0). Sections were hybridized overnight
at 37.degree. C. in a humidified chamber with a biotin-labeled
oligonucleotide probe diluted in hybridization solution (DAKO),
corresponding to the same probe as the one used for the
quantitative PCR. Probe hybridization was detected with
streptavidin/alkaline phosphatase, after addition of BCIP/NBT as a
chromogenic substrate (DAKO).
[0113] cDNA Subtractive Hybridization
[0114] Total RNA was first extracted (Williams, R. et al. J. Clin.
Invest. (2000), 106:813-818) from both ischemic and control areas
of two hearts submitted to 90-min occlusion and 1-h reperfusion.
Messenger RNA was isolated, and 2 mg was used for first-strand cDNA
synthesis with random primers. The subtractive hybridization was
performed with the PCR-select cDNA subtraction kit (CLONTECH),
following the manufacturer's recommendations. After second-strand
synthesis, the two cDNA libraries were digested with RsaI.
Digestion products of the "tester" library were ligated to a
specific adapter (T7 promoter), then hybridized with a 30-fold
excess of the "driver" library for subtraction. After
hybridization, the remaining products were further amplified by
PCR. In the forward subtraction, which determines the genes that
are over-expressed in the ischemic sample, the ischemic tissue is
the "tester" and the remote sample is the "driver." In the reverse
subtraction, the "tester" and the "driver" are switched to
determine the genes that are down-regulated in the ischemic sample.
PCR-amplified subtracted products were subcloned into the
pGEM-Teasy vector (Promega) and transformed into SURE2 cells
(Stratagene). The clones were sequenced by standard procedure
(ABI-Prizm 377 DNA sequencer, Applied Bio-systems). Sequences were
queried in public databases to determine the identity of the
genes.
[0115] Statistical Analysis
[0116] Data are expressed as mean.+-.standard deviation. The number
of samples in each experiment is indicated in the figure legends.
Statistical analysis was performed with the Student's t test. A
value of P<0.05 was considered as significant.
[0117] Results
[0118] Cloning of pDJA1
[0119] In addition to the known genes that were found in the
subtractive hybridization between stunned and normal pig myocardium
(Depre, C., et al., Gene program for cardiac cell survival induced
by transient ischemia in conscious pig, Proc. Nat'l Acad. Sci
U.S.A., (2001); 98: 9336-9341), a 0.8 Kb cDNA fragment was
subcloned which did not match any known transcript in public
databases. To determine the full-length sequence of this unknown
transcript, we screened a pig heart library with primers designed
from the 0.8 Kb fragment, and amplified the products by PCR. With
this method, 685 nucleotides of the 3'-end including a
poly-adenylation signal and the poly-A tail were obtained. Next, we
used 5'RACE PCR to obtain the remaining 5' end portion of the
transcript. Taken together, a full-length transcript corresponding
to a 3.1 Kb-long cDNA was obtained (FIGS. 1A, 1B, 1C), which is
characterized by a 62 nucleotide-long 5'-UTR, a 397 amino acid-open
reading frame and a 1.75 Kb-long 3'-UTR. The open reading frame
begins with the ATG at nucleotide 63, and is not preceded by a
Kozak's consensus for translation initiation (Kozak, M., Point
mutations define a sequence flanking the AUG initiator codon that
modulates translation by eukaryotic ribosomes, Cell, (1986); 44:
283-292). The protein has an apparent molecular weight of 44.7 KDa
and a pI=8.27. The protein contains the N-terminal J domain
characteristic of the DnaJ-like/HSP40 homologues, followed by a
glycine-rich stretch and four "zinc finger" CxxCxGxG motifs.
Interestingly, the C-terminus contains a CaaX prenylation site,
which usually characterizes proteins involved in cell growth. The
long 3'-UTR contains 7 AU-rich mRNA decay elements (Chen, C., et
al., Selective degradation of early-response genes mRNAs:
functional analyses of sequence features of the AU-rich elements,
Mol Cell Biol. (1994); 14: 8471-8482), characterized by the
sequence AUUUA. This sequence interacts with RNA-binding proteins,
which regulate the stability and half-life of transcripts usually
encoding proto-oncogenes and cytokines (Chen, C., et al., mRNA
decay mediated by two distinct AU-rich elements from c-fos and
granulocyte-macrophage colony-stimulating factor transcripts,
different deadenylation kinetics and uncoupling from translation,
Mol Cell Biol., (1995); 15: 5777-5788). The 3'UTR ends with a
poly-adenylation signal at nucleotide 2980. FIG. 2 shows the
protein sequence alignment between pDJA1 and the human HSP40. Both
proteins share a homologous N-terminus, which includes the J domain
and the G/F tract. pDJA1 totally diverges from HSP40 in its
C-terminal part, including the prenylation site which is absent in
HSP40 (FIG. 2).
[0120] Tissue Distribution of PDJA1
[0121] A pig multi-tissue Northern blot was probed, using the
original 0.8 Kb fragment of the subtractive hybridization (FIG. 3).
This Northern blot showed one specific band at 3.1 Kb,
corresponding to the full-length transcript. Remarkably, the
expression of pDJA1 was specific for the heart, as it was not
detected in the other pig tissues tested, such as stomach, kidney,
liver, lung, spleen, aorta or skeletal muscle. The distribution of
the pDJA1 transcript in myocardial tissue under baseline conditions
was further investigated and compared to the expression of other
heat-shock proteins by quantitative PCR (FIG. 4). As shown in FIG.
4A, a higher level of expression of pDJA1 was found in the
ventricles when compared to the atria, but the expression in the
left ventricle was 2-fold higher than in the right ventricle.
Interestingly, a separate analysis of subendocardial and
subepicardial samples from the left ventricle showed that the
expression of the pDJA1 transcript was double in subendocardium
over subepicardium (FIG. 4B). This distribution is specific of
pDJA1, because the transcript level of other heat-shock proteins
highly expressed in the heart, such as HSP70 and HSP40, did not
show any gradient of expression in normal left ventricle (FIG.
4C).
[0122] Upregulation of pDJA1 Transcript During
Ischemia/Reperfusion
[0123] The pDJA1 transcript was found in the subtractive library of
stunned myocardium, suggesting that this transcript is upregulated
by ischemia. To confirm this, four pig hearts were submitted to 90
minutes coronary stenosis, followed by one hour reperfusion. The
expression of pDJA1 in the ischemic area and remote area of the
same hearts was measured by Northern blot, and the signal was
normalized to the band of the 28S ribosomal RNA. As shown in FIG.
5A, the expression of pDJA1 was increased about 4-fold in the
reperfused myocardium.
[0124] To further determine the time-course of this increased
expression, additional animals were sacrificed at the end of the
90-minute occlusion period, or after 12 hours reperfusion.
Sham-operated animals, in which no coronary stenosis was performed,
were also included to test the stability of the remote area
throughout the protocol. As shown on FIG. 5B, the level of the
pDJA1 transcript slightly increased in the subendocardium during
the ischemic episode. However, a maximal and transmural increase
was observed at 1-hour reperfusion. The difference of expression
between subendocardium and subepicardium found in control hearts
persisted at all time-points during stunning. At 12 hours
reperfusion, the pDJA1 transcript returned to normal values in the
ischemic tissue (FIG. 5B). This time-course is similar to that
observed for most of the genes which are upregulated in this model
of stunning and parallels the progressive functional recovery of
stunned myocardium (Depre C., et al., Gene program for cardiac cell
survival induced by transient ischemia in conscious pig, Proc Nat'l
Acad Sci U.S.A. (2001) 98: 9336-9341). The level of the pDJA1
transcript in the remote area was similar to that in sham animals
at all time-points. We determined that this increase in pDJA1
expression was myocyte-specific by in-situ hybridization. As shown
in FIG. 5C, a strong expression was found in cardiac myocytes from
ischemic myocardium, whereas a faint signal was detected in normal
myocardium. No signal was detected in endothelial cells.
[0125] Cytoprotective Effect of PDJA1 in Isolated Cardiac
Myocytes
[0126] Adenovirus-Mediated Transfer of pDJA1
[0127] Primary cultures of ventricular cardiac myocytes were
prepared from 1-day-old Wistar rats. Cardiac myocytes were
dispersed from the ventricles by digestion with 0.1% collagenase
type IV (Worthington), 0.1% trypsin (GIBCO) and 15 .mu.g/mL DNase I
(Sigma). Cell suspensions were applied on a discontinuous Percoll
gradient (1.060/1.082 g/ml) made up in DF buffer containing
Dulbecco's Modified Eagle Medium (DMEM)/F12 (1:1, Invitrogen), 17
mM NaHCO.sub.3, 2 mM glutamine and 50 .mu.g/ml gentamycin. Cardiac
myocytes were plated on culture dishes at a density of 10.sup.6
cells per well. The culture medium was changed to a serum-free
medium after 24 hours.
[0128] The coding sequence of pDJA1 was ligated downstream of the
CMV promoter in a pDC315 shuttle vector. An adenovirus harboring
LacZ was used as a negative control. The recombinant adenoviruses
(Ade-pDJA1 and Ade-.beta.Gal) were then prepared in 293 cells by
cotransfection of a cosmid containing the adenovirus type 5 genome
(devoid of E1 and E3) with the shuttle vector, using lipofectamine
(GIBCO). Titers were determined on 293 cells overlaid with DMEM
plus 5% equine serum and 0.5% agarose. After 24 hours in culture,
cardiac myocytes were infected in serum-free medium with the
Ade-pDJA1 or the Ade.beta.Gal adenovirus. Twenty-four hours after
infection, apoptosis was induced by addition of 4 .mu.M
staurosporine (Sigma) dissolved in DMSO, and quantified by the
activation of caspase-3 (ApoTarget, BioSource).
[0129] Results
[0130] Cytoprotective Effect of pDJA1 in Isolated Cardiac
Myocytes
[0131] To confirm that pDJA1 is a co-chaperone participating in
cell survival, isolated cardiac myocytes were infected with an
adenovirus containing the coding sequence of pDJA1 under the
control of the CMV promoter (Ade-pDJA1), and compared with an
adenovirus harboring an irrelevant sequence (Ade-.beta.Gal vector).
Programmed cell death (apoptosis) was induced by addition of 4
.mu.M staurosporine for 1 hour and quantified by the measurement of
caspase-3 activation. FIG. 6 shows the increase of apoptotic rate
in presence of staurosporine as a percentage of the value found in
both groups in absence of staurosporine. After addition of
staurosporine, the stimulation of apoptosis in cells transduced
with pDJA1 was 65% lower than the values observed in the cells
transduced with the control Ade-.beta.Gal vector (FIG. 6).
Therefore, these data in vitro confirm that overexpression of pDJA1
in myocardium confers a cytoprotective effect.
Sequence CWU 1
1
713014DNASus scrofamisc_feature16n = A,T,C or G 1agacgctgcg
tttgcnggct ttgatgaaag agtgcggcgg tgccgggcgc ggagagacaa 60gatggtgaag
gagacccagt actatgacat cctgggggtg aagcccagcg cctccccgga
120ggagatcaag aaggcctatc ggaagctggc gctgaagtac cacccggaca
agaacccgga 180tgagggcgag aagtttaagc tcatatccca ggcatatgaa
gtactttcag atccaaagaa 240aagggacatt tatgaccagg gtggcgagca
ggcgattaag gaaggaggct caggcagccc 300cagcttctct tcccccatgg
acatcttcga catgttcttt ggtggcggag gacggatggc 360tagagagaga
agaggcaaga atgttgtaca tcagttgtct gtaactcttg aagatttata
420taatggagtc acaaagaaat tggctctcca gaaaaatgta atttgtgaga
aatgtgaagg 480cgttggcggg aagaagggat ctgtggagaa gtgccccgtg
tgcaaggggc gagggatgca 540gattcacatc cagcagatag ggccaggcat
ggtgcagcag atccagactg tgtgcatcga 600gtgcaagggc cagggcgagc
gcatcaaccc caaggaccgc tgcgaaaact gcagtggtgc 660caaggtcatc
cgggagaaga agatcattga ggtgcacgtg gagaaaggta tgaaagatgg
720gcaaaagata ctgtttcatg gagaaggaga tcaggagcct gagctggagc
ctggtgatgt 780cataattgtg cttgatcaga aggatcatag tgtctttcag
agacgaggcc atgacttgat 840catgaaaatg aaaattcagc tttgtgaagc
cctgtgtggc ttcaagaaga cgataaaaac 900actggatgat cgagtccttg
ttattacatc caaatcaggt gaggtgataa agcacgggga 960cctgaaatgt
gtgcgtaatg aaggaatgcc catctacaaa gcacccctgg agaaagggac
1020tctgatcata cagtttttag ttatttttcc tgaaaaacac tggcttcctc
aagacaagct 1080tccccagctg gaagctctgc tccctcctcg acagaaagtc
aggataacgg acgacatgga 1140tcaggtggag ctgaaggagt ttaatcccaa
tgagcagaac tggcgccagc acagggaggc 1200ctacgaggag gacgatgacg
ggccccgggc cggcgtgcag tgccagacgg catgaggggg 1260ccccggagca
gcatggccca gctggactag cactgatgaa tgtaaagttg gcacaatgaa
1320aatggcatcg ctttaatggc ctcgtgtttg gggtgtcctg tgtatgtgtt
cagcattctc 1380aactgctgag tgtctttttg gtttttcttt tgtttttctt
ttggttgtaa cttaagttat 1440agcttaattt atatttaaat gttttaagtg
taaatcactt ctagtctgca tatggaatct 1500gttcatttac attttcagga
aacttctgag ataccagtga ccgcactgac actttgtgct 1560tctagtggct
ttgccataat tcatttctac caataaagca cagcccagtg aacagcactt
1620agctccctag caaacctcca ggcatgaagt gggcgaactg gctcatctct
tgctccgtgc 1680ctctttgcct ccccctgccc cccatggcaa aattatgagg
gtatgatctc agggctgcta 1740atgtggcatt tccaaatcta gatgattctc
ctcaagaata aaagcacatc tgtggattgg 1800acttggctgc agggccaact
tggttcctcc tgttctgtgc ccgtgaatgt ttggaatagg 1860gtgtgagtgt
gtctgatcat ctctcttgga agcttcctga accttccaag ccttgtggtg
1920aggacaaacc agtgttttaa atgaaacgct gataaaactg tttgtgtgcg
acccctgcac 1980tgtttgttgt tttatcttct gttgacagcc tttgcagtgc
tctcccacca aagtgcttac 2040ttgtaaagaa aacgaaacca tccgtgtccc
cagcagcctc agtgcagcaa cagaagcctt 2100gggagaatgc tggtggttcg
gccccatggc acagccagct tccctgtctg accactgatc 2160ctggatgact
tgagggtctg gaaaggcaga gaacatctca gtgtttccca cctcattctc
2220ccagattcaa ctcccttcca aaggatggtt cctttccttg cacagccata
tcacaaaggg 2280cttcctgctc aagggataat gttttattta gtgagaacta
aagctctact ctggactgca 2340gtctctatag actgccatgt aaatgatagc
ttgtttgaag ggacacgagt cattaatttt 2400ctggcaggta gactacagtt
taaatttagg gctacctcaa cctttagcca ctactccttt 2460ccttcccgca
atactcacaa agaaaaattg ctgcctttct aagctgctgg gttaaagcag
2520aggccacttt tcagatacac ccttacttgg ttatacagta cctgagagtt
tgactgaggc 2580cagggacctc cccaggaggg ccaaagggca gatcagaccc
atggcaggta ggtccagagg 2640atggaccagt ctccagcaga agattgctga
ctagtgggtg ggcacaattt gcgcaaataa 2700ggtataaaaa agcctacctg
tcccactttg accaatagtc aggaaagaca taaaacctat 2760tctttcaaat
aagcctatat gaaaatcaat ttacaaatgg accacaactc cagggtgttt
2820tgtttctgtg ctgtgacttc ctaataaatt actgctagaa aattactgtc
tagttgatga 2880tggggcaaaa ttacattcag ctccttgtca tgtaatagaa
tttggagggt gttgcttgaa 2940atttatgcca cctgtacatt tgtcagctta
aaattaaaat caagctggta tgagagacaa 3000aaaaaaaaaa aaaa 30142397PRTSus
scrofa 2Met Val Lys Glu Thr Gln Tyr Tyr Asp Ile Leu Gly Val Lys Pro
Ser1 5 10 15Ala Ser Pro Glu Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ala
Leu Lys 20 25 30Tyr His Pro Asp Lys Asn Pro Asp Glu Gly Glu Lys Phe
Lys Leu Ile 35 40 45Ser Gln Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys
Arg Asp Ile Tyr 50 55 60Asp Gln Gly Gly Glu Gln Ala Ile Lys Glu Gly
Gly Ser Gly Ser Pro65 70 75 80Ser Phe Ser Ser Pro Met Asp Ile Phe
Asp Met Phe Phe Gly Gly Gly 85 90 95Gly Arg Met Ala Arg Glu Arg Arg
Gly Lys Asn Val Val His Gln Leu 100 105 110Ser Val Thr Leu Glu Asp
Leu Tyr Asn Gly Val Thr Lys Lys Leu Ala 115 120 125Leu Gln Lys Asn
Val Ile Cys Glu Lys Cys Glu Gly Val Gly Gly Lys 130 135 140Lys Gly
Ser Val Glu Lys Cys Pro Val Cys Lys Gly Arg Gly Met Gln145 150 155
160Ile His Ile Gln Gln Ile Gly Pro Gly Met Val Gln Gln Ile Gln Thr
165 170 175Val Cys Ile Glu Cys Lys Gly Gln Gly Glu Arg Ile Asn Pro
Lys Asp 180 185 190Arg Cys Glu Asn Cys Ser Gly Ala Lys Val Ile Arg
Glu Lys Lys Ile 195 200 205Ile Glu Val His Val Glu Lys Gly Met Lys
Asp Gly Gln Lys Ile Leu 210 215 220Phe His Gly Glu Gly Asp Gln Glu
Pro Glu Leu Glu Pro Gly Asp Val225 230 235 240Ile Ile Val Leu Asp
Gln Lys Asp His Ser Val Phe Gln Arg Arg Gly 245 250 255His Asp Leu
Ile Met Lys Met Lys Ile Gln Leu Cys Glu Ala Leu Cys 260 265 270Gly
Phe Lys Lys Thr Ile Lys Thr Leu Asp Asp Arg Val Leu Val Ile 275 280
285Thr Ser Lys Ser Gly Glu Val Ile Lys His Gly Asp Leu Lys Cys Val
290 295 300Arg Asn Glu Gly Met Pro Ile Tyr Lys Ala Pro Leu Glu Lys
Gly Thr305 310 315 320Leu Ile Ile Gln Phe Leu Val Ile Phe Pro Glu
Lys His Trp Leu Pro 325 330 335Gln Asp Lys Leu Pro Gln Leu Glu Ala
Leu Leu Pro Pro Arg Gln Lys 340 345 350Val Arg Ile Thr Asp Asp Met
Asp Gln Val Glu Leu Lys Glu Phe Asn 355 360 365Pro Asn Glu Gln Asn
Trp Arg Gln His Arg Glu Ala Tyr Glu Glu Asp 370 375 380Asp Asp Gly
Pro Arg Ala Gly Val Gln Cys Gln Thr Ala385 390 3953334PRTHomo
sapiens 3Met Gly Lys Asp Tyr Tyr Cys Ile Leu Gly Ile Glu Lys Gly
Ala Ser1 5 10 15Asp Glu Asp Ile Lys Lys Ala Tyr Arg Lys Gln Ala Leu
Lys Phe His 20 25 30Pro Asp Lys Asn Pro Gln Ala Glu Glu Lys Phe Lys
Glu Val Ala Glu 35 40 45Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg
Glu Ile Tyr Asp Gln 50 55 60Phe Gly Glu Glu Gly Leu Lys Gly Gly Ala
Gly Gly Asp Gly Gln Gly65 70 75 80Gly Thr Phe Arg Tyr Thr Phe His
Gly Asp Pro His Ala Thr Phe Ala 85 90 95Ala Phe Phe Gly Gly Glu Asn
Pro Phe Glu Ile Phe Phe Gly Arg Arg 100 105 110Met Gly Gly Gly Arg
Asp Ser Glu Glu Met Glu Ile Asp Gly Asp Pro 115 120 125Phe Ser Ala
Phe Gly Phe Ser Met Asn Gly Tyr Pro Arg Asp Arg Asn 130 135 140Ser
Val Gly Pro Ser Arg Leu Lys Gln Asp Pro Pro Val Ile His Glu145 150
155 160Leu Arg Val Ser Leu Glu Glu Ile Tyr Ser Gly Cys Thr Lys Arg
Met 165 170 175Lys Ile Ser Arg Lys Arg Leu Asn Ala Asp Gly Arg Ser
Tyr Arg Ser 180 185 190Glu Asp Lys Ile Leu Thr Ile Glu Ile Lys Lys
Gly Trp Lys Glu Gly 195 200 205Thr Lys Ile Thr Phe Pro Arg Glu Gly
Asp Glu Thr Pro Asn Ser Ile 210 215 220Pro Ala Asp Ile Val Pro Ile
Ile Lys Asp Lys Asp His Pro Lys Arg225 230 235 240Lys Arg Asp Gly
Ser Asn Ile Ile Tyr Thr Ala Lys Ile Ser Leu Arg 245 250 255Glu Ala
Leu Cys Gly Cys Ser Ile Asn Val Pro Thr Leu Asp Gly Arg 260 265
270Asn Ile Pro Met Ser Val Asn Asp Ile Val Lys Pro Gly Met Arg Arg
275 280 285Arg Ile Ile Gly Tyr Gly Leu Pro Phe Pro Lys Asn Pro Asp
Gln Lys 290 295 300Gly Asp Leu Leu Ile Glu Phe Glu Val Ser Phe Pro
Asp Thr Ile Ser305 310 315 320Ser Ser Ser Lys Glu Val Leu Arg Lys
His Leu Pro Ala Ser 325 3304397PRTSus scrofa 4Met Val Lys Glu Thr
Gln Tyr Tyr Asp Ile Leu Gly Val Lys Pro Ser1 5 10 15Ala Ser Pro Glu
Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys 20 25 30Tyr His Pro
Asp Lys Asn Pro Asp Glu Gly Glu Lys Phe Lys Leu Ile 35 40 45Ser Gln
Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Asp Ile Tyr 50 55 60Asp
Gln Gly Gly Glu Gln Ala Ile Lys Glu Gly Gly Ser Gly Ser Pro65 70 75
80Ser Phe Ser Ser Pro Met Asp Ile Phe Asp Met Phe Phe Gly Gly Gly
85 90 95Gly Arg Met Ala Arg Glu Arg Arg Gly Lys Asn Val Val His Gln
Leu 100 105 110Ser Val Thr Leu Glu Asp Leu Tyr Asn Gly Val Thr Lys
Lys Leu Ala 115 120 125Leu Gln Lys Asn Val Ile Cys Glu Lys Cys Glu
Gly Val Gly Gly Lys 130 135 140Lys Gly Ser Val Glu Lys Cys Pro Val
Cys Lys Gly Arg Gly Met Gln145 150 155 160Ile His Ile Gln Gln Ile
Gly Pro Gly Met Val Gln Gln Ile Gln Thr 165 170 175Val Cys Ile Glu
Cys Lys Gly Gln Gly Glu Arg Ile Asn Pro Lys Asp 180 185 190Arg Cys
Glu Asn Cys Ser Cys Ala Lys Val Ile Arg Glu Lys Lys Ile 195 200
205Ile Glu Val His Val Glu Lys Cys Met Lys Asp Gly Gln Lys Ile Leu
210 215 220Phe His Gly Glu Cys Asp Gln Glu Pro Glu Leu Glu Pro Gly
Asp Val225 230 235 240Ile Ile Val Leu Asp Gln Lys Asp His Ser Val
Phe Gln Arg Arg Gly 245 250 255His Asp Leu Ile Met Lys Met Lys Ile
Gln Leu Cys Glu Ala Leu Cys 260 265 270Gly Phe Lys Lys Thr Ile Lys
Thr Leu Asp Asp Arg Val Leu Val Ile 275 280 285Thr Ser Lys Ser Gly
Glu Val Ile Lys His Gly Asp Leu Lys Cys Val 290 295 300Arg Asn Glu
Gly Met Pro Ile Tyr Lys Ala Pro Leu Glu Lys Gly Thr305 310 315
320Leu Ile Ile Pro Val Leu Val Val Phe Pro Arg Lys His Trp Leu Pro
325 330 335Gln Asp Lys Leu Pro Gln Leu Glu Ala Leu Leu Pro Pro Arg
Gln Lys 340 345 350Val Arg Ile Thr Asp Asp Met Asp Gln Val Glu Leu
Lys Glu Phe Asn 355 360 365Pro Asn Glu Gln Asn Trp Arg Gln His Arg
Glu Ala Tyr Glu Glu Asp 370 375 380Asp Asp Gly Pro Arg Ala Gly Val
Gln Cys Gln Thr Ala385 390 395522DNAArtificial SequencePrimer
5ctctcttgga agcttcctga ac 22619DNAArtificial SequencePrimer
6gcactgcaaa ggctgtcaa 19729DNAArtificial SequenceProbe 7aagcttgtgg
tgaggacaaa ccagtgttt 29
* * * * *
References