Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
2002-02-19
2004-12-21
Martinell, James (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C514S012200, C424S185100
Reexamination Certificate
active
06833435
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates generally to polypeptides expressed in bovine tissues, including mammary gland cells and paratoid salivary gland cells, to polynucleotides encoding such polypeptides, and to methods for treating a mammal involving administration of a polypeptide or polynucleotide of the present invention. More particularly, the present invention relates to polynucleotides that encode or statherins and/or histatins, together with polypeptides encoded by such polynucleotides and methods for the use of such polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
Human histatins are a family of low molecular weight (51-77 amino acids), neutral to very basic, histidine-rich, proteins that are specific to salivary secretions. Histatins are believed to function as part of the non-immune defense system, particularly in the oral cavity. Salivary histatins have been shown to be potent antifungal, antimicrobial and antibacterial agents, and to have promise as therapeutic agents in humans with oral candidosis (Tsai et al.
Infection and Immunity
64:5000-5007, 1996). In particular, histatins have been shown to kill the pathogenic yeast,
Candida albicans
(Oppenheim et al.
J. Biol. Chem
. 263:7472-7477, 1988), with peptides representing defined portions of the amino acid sequences of naturally occurring human and macaque histatin being shown to have superior anti-candidal activity over the intact native histatin (Zuo et al.
Gene
161:87-91, 1995; U.S. Pat. No. 5,486,503).
Human statherin is a low molecular weight, acidic phosphoprotein that acts to inhibit precipitation of calcium phosphate salts in the oral cavity, and which is believed to belong to the same gene family as human histatins (Sabatini et al.
Mol. Biol. Evol
. 10:497-511, 1993).
Fungal and bacterial infections are common and can be life threatening in patients with compromised immune systems. Candidal infections often occur in diabetics, during pregnancy and following medication with antibiotics, steroid hormones or oral contraceptives. Oral candidiasis is common in patients infected with HIV, as well as in cancer patients following treatment with radiation or chemotherapy. Systemic fungal infections in immuno-compromised patients and in patients in intensive care are often fatal since there are few effective anti-fungal treatments for intravenous administration. Similarly, bacterial infections can lead to severe disease and even death. Although several anti-fungal and ant-bacterial agents are available, these agents are not completely effective, with many not being appropriate for oral or systemic administration. Thus there remains a need in the art for preparations have effective anti-fungal and anti-bacterial properties.
SUMMARY OF THE INVENTION
The present invention provides isolated histatin-like polypeptides expressed in bovine tissues and isolated polynucleotides encoding such polypeptides, together with genetic constructs and host cells comprising such polynucleotides. Methods for using such polypeptides, polynucleotides and genetic constructs are also provided.
In specific embodiments, isolated polynucleotides are provided that comprise a polynucleotide sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-6; (b) complements of the sequences recited in SEQ ID NOS: 1-6; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-6; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-6; and (e) sequences having at least 75%, 90% or 95% identity to a sequence of (a)-(d), the percentage identity being determined as described below. Polynucleotides comprising at least a specified number of contiguous residues (“x-mers”) of any of the sequences identified as SEQ ID NOS: 1-6 are also provided, together with extended sequences, and oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-6. All of these polynucleotides and oligonucleotide probes and primers are collectively referred to herein as “polynucleotides of the present invention”.
In further embodiments, the present invention provides isolated polypeptides comprising an amino acid sequence encoded by a polynucleotide selected from the group consisting of: (a) sequences provided in SEQ ID NOS: 1-6; and (b) sequences having at least 75%, 90% or 95% identity to a sequence provided in SEQ ID NOS: 1-6. In specific embodiments, such polypeptides comprise a sequence selected from the group consisting of sequences identified as SEQ ID NO: 7-12, and variants thereof. The present invention further provides isolated polypeptides comprising at least a functional portion of an amino acid sequence encoded by a polynucleotide selected from the group consisting of: (a) sequences provided in SEQ ID NOS: 1-6; and (b) sequences having 75%, 90% or 95% identity to a sequence of SEQ ID NOS: 1-6.
In related embodiments, the present invention provides genetic constructs comprising the inventive polynucleotides, together with host cells transformed with such constructs, and organisms comprising such host cells.
In a further aspect, the present invention provides compositions comprising an inventive polypeptide or polynucleotides in combination with a physiologically acceptable carrier and/or an immunostimulant, together with methods for the use of such compositions in the treatment of a disorder in a mammal, such as a cow or a human. Preferably the disorder is selected from the group consisting of bacterial, microbial and fungal infections. Such infections may be either local or systemic. In certain embodiments, anti-microbial cosmetic compositions comprising the inventive polypeptides are provided.
The isolated polynucleotides of the present invention have further utility in genome mapping, in physical mapping, and in positional cloning of genes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-6, and their variants, may be used to design oligonucleotide probes and primers (referred to collectively as “oligonucleotides”). As detailed below, oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. The inventive oligonucleotide probes may be used to detect the presence, and examine the expression patterns, of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot blot DNA hybridization techniques. The inventive oligonucleotide primers may be used for PCR amplifications. Oligonucleotide probes and primers of the present invention may also be used in connection with various microarray technologies, including the microarray technology of Affymetrix, Inc. (Santa Clara, Calif.).
The above-mentioned and additional features of the present invention, together with the manner of obtaining them, will be best understood by reference to the following more detailed description. All references disclosed herein are incorporated herein by reference in their entirety as if each was incorporated individually.
DETAILED DESCRIPTION OF THE INVENTION
In certain aspects, the present invention provides polynucleotides that were isolated by sequencing of cDNA libraries from bovine tissues, including mammary gland cells and paratoid salivary gland cells, together with isolated polypeptides encoded by such polynucleotides. The polynucleotides of the present invention encode polypeptides that show similarities to both histatins and statherins. As discussed above, histatins have been shown to possess toxic activity against a large range of organisms, including oral bacterial and
Candida albicans
. Accordingly, the inventive polypeptides may be employed in the treatment of fungal and bacterial infections of the oral cavity, vagina, urethra, ear, skin, respiratory tract, mucosa and eye, as well in the treatment of systemic infections.
Organisms against which the inventive compositions may be effectively employed include, but are not limited to:
Candida albicans
(bot
Davis Stephen R.
Glenn Matthew
Grigor Murray R.
Molenaar Adrian J.
Friedman Susan J.
Martinell James
Sleath Janet
Speckman Ann W.
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