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Patent application title: ANTIBIOTIC SYNERGISM
Per Holse Mygind (Vaerloese, DK)
Per Holse Mygind (Vaerloese, DK)
Dorthe Sandvang (Slangerup, DK)
Hans-Henrik Kristensen Hoegenhaug (Holte, DK)
Thomas Kruse (Copenhagen, DK)
Line Anker Nielsen (Copenhagen, DK)
Novozymes A/S
IPC8 Class: AA61K3800FI
USPC Class:
Class name: Drug, bio-affecting and body treating compositions plural fermentates of different origin (e.g., mixtures of antibiotics, etc.)
Publication date: 2010-04-29
Patent application number: 20100104552
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Patent application title: ANTIBIOTIC SYNERGISM
Hans-Henrik Kristensen Hoegenhaug
Dorthe Sandvang
Per Holse Mygind
Thomas Kruse
Line Anker Nielsen
Novozymes A/S
Origin: NEW YORK, NY US
IPC8 Class: AA61K3800FI
USPC Class:
Patent application number: 20100104552
The present invention relates to antibiotic synergism of pharmaceutical
compositions comprising a defensin and a beta-lactam antibiotic.
1. A pharmaceutical composition comprising:(a) a first antibacterial
compound which is a defensin, and(b) a second antibacterial compound
which is a beta-lactam antibiotic or an aminoglycoside.
2. The composition of claim 1, wherein the first and second antibacterial compound exhibit a synergistic antibacterial activity.
3. The composition of claim 2, wherein the first and second antibacterial compound exhibit a FIC index ≦0.5 using a test organism selected from the group consisting of Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC29213, Staphylococcus aureus ATCC34400, Staphylococcus aureus ATCC25923, and Staphylococcus epidermidis ATCC49134.
4. The composition of claim 1, wherein the first antibacterial compound binds to the bacterial cell wall precursor Lipid I and/or Lipid II.
5. The composition of claim 4, wherein the binding results in sequestering of Lipid I and/or Lipid II.
6. The composition of claim 4, wherein the binding is characterized by a binding constant of about 10.sup.-6 M-1 or less, preferably by a binding constant of about 10.sup.-7 M.sup.-1.
7. The composition of claim 1, wherein the first antibacterial compound belongs to the arthropod defensin class, or the insect defensin class.
8. The composition of claim 1, wherein the defensin comprises an amino acid sequence having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably at least 97% identity to the amino acid sequence of SEQ ID NO: 1.
9. The composition of claim 1, wherein the second antibacterial compound is selected from the group consisting of penicillins, cephalosporins and aminoglycosides.
10. The composition of claim 1, wherein the second antibacterial compound is selected from the group consisting of Penicillin G, Ceftriaxone and Gentamicin.
11. A method for the treatment of an intracellular bacterial infection, comprising administering to a human or animal in need of such treatment a composition of claim 1 in an effective amount for the treatment of the intracellular bacterial infection.
12. The method of claim 11, wherein the bacterial infection is caused by Gram-positive bacteria.
13. The method of claim 12, wherein the bacterial infection is caused by Streptococci or Staphylococci.
[0001]This application claims priority or the benefit under 35 U.S.C. 119 of European application nos. EP 08167451.7, EP 08170260.7, and EP 09153967.6 filed Oct. 23, 2008, Nov. 28, 2008 and Feb. 27, 2009, respectively, and U.S. provisional application NOS. 61/112,358, U.S. 61/118,782 and 61/157,630 filed Nov. 7, 2008, Dec. 1, 2008 and Mar. 5, 2009, respectively, the contents of which are fully incorporated herein by reference.
[0002]This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
[0003]1. Field of the Invention
[0004]The present invention relates to synergism between defensin antibiotics and beta-lactam antibiotics.
[0005]2. Description of Related Art
[0006]The few available distinct classes of antimicrobial compounds limit the scope for single and combination drug treatment of bacterial infections, including infections involving antibiotic-resistant bacteria. Antibacterial chemotherapy research has therefore focused on the discovery of novel targets for new antibacterial development. An alternative approach to the discovery of new antibacterial compounds is the discovery of antibiotic synergists. Synergism in antimicrobial therapy is well known and is used to describe supra-additive activity of antibiotics used in combinations. For example, in the treatment of bacterial infections combinations such as penicillin or ampicillin and streptomycin or gentamycin have been shown to have a supra-additive effect against enterococci infections. Similarly, carbenicillin or ticarcillin combined with an aminoglycoside such as gentamycin or tobramycin exhibit a synergistic effect in the treatment of Pseudomonas aeruginosa infection. Combined therapy using streptomycin together with tetracycline is more effective in the therapy of brucellosis than either agent alone, and a mixture of chloramphenicol plus a sulfonamide is more effective against meningitis due to Haemophilus influenzae.
[0007]Several antibacterial defensin polypeptides are known in the art. Examples include Plectasin (see WO 03/044049) and variants of Plectasin (see WO 2006/131504). Beta-lactam antibiotics are also a well-known group of antibacterial compounds. A beta-lactam (13-lactam) is a lactam with a heteroatomic ring structure, consisting of three carbon atoms and one nitrogen atom. The beta-lactam ring is part of several antibiotics, such as penicillin, which are therefore also called beta-lactam antibiotics.
[0008]Combinations of defensins and beta-lactam antibiotics, and therapeutic uses thereof, have not previously been disclosed.
[0009]It is an object of the present invention to provide a synergistic combination therapy of using defensins and beta-lactam antibiotics.
[0010]We have now found that defensin polypeptides and beta-lactam antibiotics exhibit synergistic antibacterial activity.
[0011]Accordingly, the present invention provides a (synergistic) pharmaceutical composition comprising a first antibacterial compound which is a defensin, and a second antibacterial compound which is a beta-lactam antibiotic or an aminoglycoside.
[0012]In a second aspect, the invention provides a method for the treatment of a bacterial infection in a human or animal, comprising administering to the human or animal in need of such treatment a first antibacterial compound which is a defensin, and a second antibacterial compound which is a beta-lactam antibiotic or an aminoglycoside, in an effective amount for the treatment of the bacterial infection.
[0013]Antibacterial activity: The term "antibacterial activity" is defined herein as an activity which is capable of killing or inhibiting growth of bacterial cells. In the context of the present invention the term "antibacterial" is intended to mean that there is a bactericidal and/or a bacteriostatic effect; wherein the term "bactericidal" is to be understood as capable of killing bacterial cells; and wherein the term "bacteriostatic" is to be understood as capable of inhibiting bacterial growth. When growth of bacterial cells is inhibited, the cells are in a non-growing state, i.e., they are not able to propagate.
[0014]In a preferred embodiment, the term "antibacterial activity" is defined as bactericidal and/or bacteriostatic activity against Streptococci, preferably Streptococcus pneumoniae, or Staphylococci, preferably Staphylococcus aureus.
[0015]For purposes of the present invention, antibacterial activity may be determined according to the procedure described by Lehrer et al., 1991, Journal of Immunological Methods 137(2): 167-174. Alternatively, antibacterial activity may be determined according to the NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards).
[0016]Compounds having antibacterial activity may be capable of reducing the number of living cells of Streptococcus pneumoniae (ATCC 49619) to 1/100 after 24 hours (preferably after 16 hours, more preferably after 8 hours, most preferably after 4 hour, and in particular after 2 hours) incubation at 37° C. in a relevant microbial growth substrate at a concentration of 500 micrograms/mL; preferably at a concentration of 250 micrograms/mL; more preferably at a concentration of 100 micrograms/mL; even more preferably at a concentration of 50 micrograms/mL; most preferably at a concentration of 25 micrograms/mL; and in particular at a concentration of 10 micrograms/mL of the polypeptides having antimicrobial activity.
[0017]Compounds having antibacterial activity may also be capable of inhibiting the outgrowth of Streptococcus pneumoniae (ATCC 49619) for 8 hours at 37° C. in a relevant microbial growth substrate, when added in a concentration of 500 micrograms/mL; preferably when added in a concentration of 250 micrograms/mL; more preferably when added in a concentration of 100 micrograms/mL; even more preferably when added in a concentration of 50 micrograms/mL; most preferably when added in a concentration of 10 micrograms/mL; and in particular when added in a concentration of 5 micrograms/mL.
[0018]The compounds of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the antibacterial activity of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
[0019]Defensin: The term "defensin" as used herein refers to polypeptides recognized by a person skilled in the art as belonging to the defensin class of antimicrobial peptides. To determine if a polypeptide is a defensin according to the invention, the amino acid sequence is preferably compared with the hidden markov model profiles (HMM profiles) of the PFAM database by using the freely available HMMER software package (see Example 5).
[0020]The PFAM defensin families include Defensin--1 or "Mammalian defensin" (accession no. PF00323), Defensin--2 or "Arthropod defensin" (accession no. PF01097), Defensin_beta or "Beta Defensin" (accession no. PF00711), Defensin_propep or "Defensin propeptide" (accession no. PF00879) and Gamma-thionin or "Gamma-thionins family" (accession no. PF00304).
[0021]The defensins may belong to the alpha-defensin class, the beta-defensin class, the theta-defensin class, the insect or arthropod defensin classes, or the plant defensin class. The defensins from each of these classes share common structural features, such as the cysteine pattern. But it is important to note that the class affiliation does not reveal the source of the defensins. For example, a defensin from a fungus may be affiliated to the insect defensin class.
[0022]The defensin shown as SEQ ID NO: 1 is a synthetic defensin derived from Plectasin (see WO 03/044049), which is affiliated with the insect defensin class.
[0023]In an embodiment, the amino acid sequence of a defensin according to the invention comprises 4, 5, 6, 7, or 8 cysteine residues, preferably 4, 5, or 6 cysteine residues, more preferably 4 or 6 cysteine residues, and most preferably 6 cysteine residues.
[0024]The defensins may also be synthetic defensins sharing the characteristic features of any of the defensin classes.
[0025]Examples of such defensins include, but are not limited to, a-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; β-Defensin-12, Drosomycin, Heliomicin, γ1-purothionin, Insect defensin A, and the defensins disclosed in PCT applications WO 99/53053, WO 02/06324, WO 02/085934, WO 03/044049, WO 2006/131504, WO 2006/050737 and WO 2006/053565.
[0026]Isolated polypeptide: The term "isolated variant" or "isolated polypeptide" as used herein refers to a variant or a polypeptide that is isolated from a source. In one aspect, the variant or polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.
[0027]Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.
[0028]Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".
[0029]For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the--nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment-Total Number of Gaps in Alignment)
[0030]For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra; emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the--nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment-Total Number of Gaps in Alignment).
Allelic variant: The term "allelic variant" denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0031]Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 as well as genetic manipulation of the DNA encoding that polypeptide. The modification(s) can be substitution(s), deletion(s) and/or insertions(s) of the amino acid(s) as well as replacement(s) of amino acid side chain(s); or use of unnatural amino acids with similar characteristics in the amino acid sequence. In particular the modification(s) can be amidations, such as amidation of the C-terminus.
Defensin Polypeptides Having Antibacterial Activity
[0032]In a first aspect, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to SEQ ID NO: 1 (i.e., the mature polypeptides) of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%, and in particular at least 97%, which have antibacterial activity (hereinafter "homologous polypeptides").
[0033]In a preferred aspect, the homologous polypeptides have an amino acid sequence which differs by at the most six amino acids, preferably by at the most five amino acids, more preferably by at the most four amino acids, even more preferably by at the most three amino acids, most preferably by at the most two amino acids, and in particular by one amino acid from the amino acid sequence of SEQ ID NO: 1.
[0034]In another preferred aspect, the homologous polypeptides have an amino acid sequence which differs by at the most six amino acids, preferably by at the most five amino acids, more preferably by at the most four amino acids, even more preferably by at the most three amino acids, most preferably by at the most two amino acids, and in particular by one amino acid from the amino acid sequence of SEQ ID NO: 2.
[0035]The amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 have three amino acid differences, which are in positions 9, 13 and 14.
[0036]A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof. In a preferred aspect, a polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In another preferred aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof. In another preferred aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
[0037]Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the polypeptide; single deletions; small amino- or carboxyl-terminal extensions; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tag, an antigenic epitope or a binding domain.
[0038]Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0039]In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.
[0040]Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., antimicrobial activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention.
[0041]Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).
[0042]Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
N-Terminal Extension
[0043]An N-terminal extension of the polypeptides of the invention may suitably consist of from 1 to 50 amino acids, preferably 2-20 amino acids, especially 3-15 amino acids. In one embodiment N-terminal peptide extension does not contain an Arg (R). In another embodiment the N-terminal extension comprises a kex2 or kex2-like cleavage site as will be defined further below. In a preferred embodiment the N-terminal extension is a peptide, comprising at least two Glu (E) and/or Asp (D) amino acid residues, such as an N-terminal extension comprising one of the following sequences: EAE, EE, DE and DD.
Kex2 Sites
[0044]Kex2 sites (see, e.g., Methods in Enzymology, Vol 185, ed. D. Goeddel, Academic Press Inc. (1990), San Diego, Calif., "Gene Expression Technology") and kex2-like sites are di-basic recognition sites (i.e., cleavage sites) found between the pro-peptide encoding region and the mature region of some proteins.
[0045]Insertion of a kex2 site or a kex2-like site have in certain cases been shown to improve correct endopeptidase processing at the pro-peptide cleavage site resulting in increased protein secretion levels.
[0046]In the context of the invention insertion of a kex2 or kex2-like site result in the possibility to obtain cleavage at a certain position in the N-terminal extension resulting in an antimicrobial polypeptide being extended in comparison to the mature polypeptide shown in SEQ ID NO: 1.
Fused Polypeptides
[0047]The polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the invention or a fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
Mechanism-Of-Action of the Defensins Shown as SEQ ID NO: 1 and SEQ ID NO: 2
[0048]The mechanism by which the defensins shown as SEQ ID NO: 1 (hereinafter "Defensin2114") and SEQ ID NO: 2 (hereinafter "Plectasin") kills bacterial cells has been thoroughly investigated using a number of techniques. In contrast to many other antimicrobial peptides, Defensin2114 and Plectasin do not initially and as a main mechanism compromise the bacterial membrane. No K+-efflux could be detected and the trans-membrane potential of bacteria exposed to Defensin2114 or Plectasin remained intact.
[0049]Traditional incorporation studies showed that exposure to Defensin2114 or Plectasin severely affected incorporation of cell wall precursors but not those of proteins or nucleic acids (RNA/DNA).
[0050]It has been shown in complementing experiments such as DNA microarray, growth kinetics and changes in cellular morphology that the result of Defensin2114-exposure or Plectasin-exposure mimics the response after exposure to other cell-wall inhibiting agents and mostly that of vancomycin. More specifically, it has been shown in vitro that Defensin2114 and Plectasin binds to LipidI and LipidII--precursors used to synthesize the bacterial cell wall--with binding-constants in the 10-6 to 10-7 M-1 range. Sequestering of LipidII (and LipidI) in the bacterial cell inhibits the polymerization of these precursors into peptidoglycan. The polymerization of LipidI into peptidoglycan is a result of an initial trans-glycosylation of the sugar-moieties followed by a transpeptidase reaction where the cross-linking of different sugar-strands is mediated through the peptide part of LipidII. Both reactions could in principle be blocked by Defensin2114 or Plectasin.
[0051]Other antibiotics and antimicrobials have also been shown to bind and sequester LipidII, e.g., the glycopeptide vancomycin and the Iantibiotic Nisin. No cross-resistance have been observed between, e.g., vancomycin-resistant Staphylococcus aureus (VRSA) and Defensin2114 or Plectasin indicating that different epitopes on LipidII is involved in/important for binding of these two antibiotics.
Mechanism-Of-Action of Beta-Lactam Antibiotics
[0052]The beta-lactam ring is part of the structure of several antibiotic families, principally the penicillins, cephalosporins, carbapenems and monobactams, which are therefore also called beta-lactam antibiotics. These antibiotics work by inhibiting the bacterial cell wall synthesis. Beta-lactam antibiotics (such as Penicillin G) work by inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall. The β-lactam moiety (functional group) of penicillin binds to the enzyme (DD-transpeptidase) that links the peptidoglycan molecules in bacteria, which weakens the cell wall of the bacterium (in other words, the antibiotic causes cytolysis or death due to osmotic pressure). In addition, the build-up of peptidoglycan precursors triggers the activation of bacterial cell wall hydrolases and autolysins, which further digest the bacteria's existing peptidoglycan.
[0053]Penicillin shows a synergistic effect with aminoglycosides (such as gentamicin), since the inhibition of peptidoglycan synthesis allows aminoglycosides to penetrate the bacterial cell wall more easily, allowing its disruption of bacterial protein synthesis within the cell. This results in a lowered MBC for susceptible organisms.
[0054]Cephalosporin compounds were first isolated from cultures of Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist Giuseppe Brotzu. The cephalosporin nucleus, 7-aminocephalosporanic acid (7-ACA), was derived from cephalosporin C and proved to be analogous to the penicillin nucleus 6-aminopenicillanic acid. Therefore, cephalosporin compounds belong to the group of beta-lactam antibiotics.
[0055]Cephalosporins (such as Ceftriaxone or Cefotaxime) are bactericidal and have the same mode of action as other beta-lactam antibiotics (such as penicillins). Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to crosslink the peptidoglycan. Beta-lactam antibiotics mimic this site and competitively inhibit PBP crosslinking of peptidoglycan.
Mechanism of Action of Aminoglycosides
[0056]Aminoglycosides (such as Gentamicin) work by binding to the bacterial 30S ribosomal subunit (some work by binding to the 50S subunit), inhibiting the translocation of the peptidyl-tRNA from the A-site to the P-site and also causing misreading of mRNA, leaving the bacterium unable to synthesize proteins vital to its growth. They kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane.
Compositions Exhibiting Synergistic Antibacterial Activity
[0057]The present invention provides a pharmaceutical composition, exhibiting synergistic antibacterial activity, comprising: [0058]a first antibacterial compound which is a defensin, and [0059]a second antibacterial compound which is a beta-lactam antibiotic or an aminoglycoside.
[0060]Synergism is identified in two-dimensional or checkerboard tests when the Fractional Inhibitory Concentration (FIC) index is ≦0.5. In killing curves, synergy is identified as a ≧2 Log10, decrease in CFU/mL between the combination and the most active constituent after 24 hours; and the number of surviving organisms in the presence of the combination must be ≧2 Log10 CFU/mL below the starting inoculum. At least one of the drugs must be present in a concentration which does not affect the growth curve of the test organism, when used alone. Antagonism is identified by a FIC index ≧4. See also Examples 1 and 2.
[0061]In an embodiment, the composition of the invention exhibits a FIC index ≦0.5 using a test organism selected from the group consisting of Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC29213, Staphylococcus aureus ATCC34400, Staphylococcus aureus ATCC25923, and Staphylococcus epidermidis ATCC49134.
[0062]In another embodiment, the composition of the invention exhibits synergistic antibacterial activity on Gram-positive bacteria, such as streptococci and staphylococci.
Methods and Uses
[0063]The composition of the invention is used for treating bacterial infections. Accordingly, the composition of the invention may be used as an antibacterial veterinarian or human therapeutic or prophylactic agent. The composition of the invention may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of bacterial infections.
[0064]The composition of the invention is used in an amount sufficient to treat the bacterial infection, i.e., sufficient to kill or inhibit the bacteria causing the bacterial infection; for example, to kill or inhibit growth of Streptococcus sp., such as Streptococcus pneumoniae, or Staphylococcus sp., such as Staphylococcus aureus.
[0065]Formulations of the composition of the invention are administered to a host suffering from a bacterial infection. Administration may be localized or systemic. Generally the dose of the composition of the invention will be sufficient to decrease the bacterial population by at least 1 log, and may be by 2 or more logs of killing. The composition of the invention is administered at a dosage that reduces the bacterial population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use.
[0066]The antibacterial compounds used in the composition of the invention may be administered simultaneously, or after each other, in any order. The antibacterial compounds may also be administered independently of each other via different routes. The following examples of administration and formulation apply to the composition of the invention, as well as to the individual antibacterial compounds used in the composition.
[0067]Various methods for administration may be employed. The formulation of the antibacterial compounds may be given orally, or may be injected intravascularly, intramuscular, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. The dosage of the therapeutic formulation will vary widely, depending on the specific antibacterial compounds to be administered, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds of the defensin, as well as the amino and carboxy termini, may be modified for greater stability on oral administration. For example, the carboxy terminus may be amidated.
[0068]The antibacterial compounds used in the composition of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the antibacterial compounds can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the antibacterial compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The antibacterial compounds of the composition of the invention may be systemic after administration or may be localized.
[0069]The composition of the present invention can be administered alone, or in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.). In pharmaceutical dosage forms, the antibacterial compounds of the composition may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely examples and are in no way limiting.
[0070]For oral preparations, the antibacterial compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
[0071]The antibacterial compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
[0072]The antibacterial compounds can be utilized in aerosol formulation to be administered via inhalation. The antibacterial compounds used in the composition of the invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
[0073]Furthermore, the antibacterial compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The antibacterial compounds can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
[0074]Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the antibacterial compounds used in the composition of the invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the antibacterial compounds as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
[0075]Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing the composition of the invention is placed in proximity to the site of infection, so that the local concentration of the antibacterial compounds is increased relative to the rest of the body.
[0076]The term "unit dosage form", as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of antibacterial compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular antibacterial compounds employed and the effect to be achieved, and the pharmacodynamics associated with the antibacterial compounds in the host.
[0077]The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
[0078]Typical dosages for systemic administration range from 0.1 pg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
[0079]Those of skill will readily appreciate that dose levels can vary as a function of the specific antibacterial compounds, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific antibacterial compounds are more potent than others. Preferred dosages for a given antibacterial compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given antibacterial compound.
[0080]The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will normally be neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
[0081]For preparing the liposomes, the procedure described by Kato et al., 1991, J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing antibacterial compounds are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 sec., the tube is placed in a warm water bath, from about 25-40° C. and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules.
Formulations with Other Active Agents
[0082]For use in the subject methods, the antibacterial compounds used in the composition of the invention may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with beta-lactamase inhibitors, cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.
[0083]Anti-mycotic agents are also useful, including polyenes, e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol and fluconazol. Antituberculotic drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in a formulation of the antibacterial compounds, e.g., interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.
In vitro Synthesis
[0084]The defensin polypeptides used in the composition of the invention may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms), e.g., D-alanine and D-isoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
[0085]Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g., reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like.
[0086]If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
[0087]The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein
[0088]The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
[0089]The defensin polypeptide shown as SEQ ID NO: 1 is hereinafter referred to as "Defensin2114".
[0090]The defensin polypeptide shown as SEQ ID NO: 2 is hereinafter referred to as "Plectasin".
Example 1
Antibiotic Synergism I
[0091]Synergy, additivity and antagonism between Defensin2114 and a variety of clinically employed antibiotics were investigated using a Time-Kill approach against Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC29213, Staphylococcus aureus ATCC34400, Staphylococcus aureus ATCC25923, and Staphylococcus epidermidis ATCC49134.
[0092]In this approach, individual antibiotics at 1/2×MIC were added to 107 CFU/mL staphylococcal or streptococcal cells, either alone or in specific combinations, and the CFU was determined at 2, 3 and 5 hours after exposure.
[0093]Synergy was defined as a ≧2 log10 decrease in CFU/ml for the combination compared with the most active single agent alone.
[0094]Additivity was defined as a ≧1 log10 decrease with the combination in comparison with the most active single antimicrobial alone.
[0095]Antagonism was defined as a ≧2 log10 increase in colony count. The antibiotics tested in combination with Defensin2114 were: Gentamicin, Penicillin G, Ceftriaxone, Vancomycin, Erythromycin, Ciprofloxacin, Tetracycline, Polymyxin B, Fucidin, Linezolid, Mupirocin and Choramphenicol.
[0096]Synergism was identified between Defensin2114 and Penicillin G, and between Defensin2114 and Ceftriaxone. Both Penicillin G and Ceftriaxone are beta-lactam antibiotic compounds that inhibit the transpeptidation step in the synthesis of the bacterial peptidoglycan. Defensin2114 and Gentamicin showed synergy at some but not all time-points.
[0097]Lack of antagonism was observed between Defensin2114 and all compounds tested (Gentamicin, Penicillin G, Ceftriaxone, Vancomycin, Erythromycin, Ciprofloxacin, Tetracycline, Polymyxin B, Fucidin, Linezolid, Mupirocin and Choramphenicol).
Example 2
Antibiotic Synergism II
[0098]Synergy, additivity and antagonism between Defensin2114 and a variety of clinically employed antibiotics was also investigated using a checkerboard titration assay against Staphylococcus aureus ATCC29213.
[0099]This approach can be described essentially as an MIC/MBC assay in 2 dimensions. In short, one antibiotic is serially 2-fold diluted along one direction of a microtiter plate while the second antibiotic is diluted along the other direction of the microtiter plate. In addition, each well contains a suspension of ˜5×104 bacteria, and the plate is read after 18-24 hours of incubation at 37° C. The resulting growth or no growth is recorded and the Fractional Inhibitory Concentration (FIC) index is calculated as the minimal combination of the two compounds that inhibits growth on the microtiter plate following the formula below:
FIC index=(conc. of drug A/MIC of drug A)+(conc. of drug B/MIC of drug B)
[0100]Fractional Bactericidal Concentration (FBC) is calculated in a similar manner, but based on MBC.
[0101]Interpretation of results is (in accordance with the literature):
TABLE-US-00001 FBC/FIC Index ≦0.5 ~synergism 0.5 < FBC/FIC Index <4 ~lack of synergy/antagonism FBC/FIC Index ≧4.0 ~antagonism
[0102]The antibiotics tested in combination with Defensin2114 included: Erythromycin, Penicillin G, Gentamicin, Ceftriaxone, Vancomycin, Ciprofloxacin and Linezolid.
[0103]The calculated FIC/FBC indices are shown in Table 1.
TABLE-US-00002 TABLE 1 FIC FBC First antibiotic Second antibiotic index index Defensin2114 Defensin2114 1 -- Defensin2114 Erythromycin 1.5 -- Defensin2114 Penicillin G 0.5 0.5 Defensin2114 Gentamicin 1 -- Defensin2114 Ceftriaxone 0.5 -- Defensin2114 Vancomycin 2 -- Defensin2114 Ciprofloxacin 1 -- Defensin2114 Linezolid 2 --
[0104]As observed in the Time-Kill approach described in Example 1, synergism between Defensin2114 and Penicillin G or Ceftriaxone (FBC/FIC=0.5) was found against Staphylococcus aureus.
[0105]No FIC antagonism was found between Defensin2114 and Erythromycin, Gentamicin, Vancomycin, Ciprofloxacin and Linezolid.
Example 3
Antibiotic Synergism III
[0106]Synergy between Defensin2114 and clinically employed antibiotics, penicillin-G and ceftriaxone, was investigated using a checkerboard titration assay against representative species of Entorococci, Staphylococci and Streptococci. This approach can be described essentially as a MIC assay in two dimensions. In short, one antibiotic is serially two-fold diluted along one direction of the microtitre plate while the second antibiotic is diluted along the other direction. In addition, each well contains a suspension of ˜5×104 bacteria and the plate is read after 18 hours of incubation at 37° C. The analysis results in a checkerboard of serial dilutions of both antibiotics. The Fractional Inhibitory Concentration (FIC) index is generally calculated as the minimal combination of the two compounds that inhibits growth on the microtitre plate. This value was calculated as:
FIC index=(Conc. of drug A/MIC of drug A)+(Conc. of drug B/MIC of drug B)
[0107]Interpretation of results was (in accordance with the literature):
TABLE-US-00003 FBC/FIC Index ≦0.5 ~synergism 0.5 < FBC/FIC Index <4 ~lack of synergy/antagonism FBC/FIC Index ≧4.0 ~antagonism
[0108]The study resulted in FIC indices indicating antibiotic synergy of the following combinations of antibiotics, as described in Table 2 below.
TABLE-US-00004 TABLE 2 FIC 1. antibiotic 2. antibiotic Target strain ATCC# index Defensin2114 Ceftriaxone Enterococcus faecalis 29212 0.25 Defensin2114 Penicillin G Staphylococcus aureus 25923 0.5 Defensin2114 Ceftriaxone Staphylococcus aureus 25923 0.5 Defensin2114 Penicillin G Staphylococcus aureus 29213 0.5 Defensin2114 Ceftriaxone Staphylococcus aureus 29213 0.5 Defensin2114 Penicillin G Staphylococcus aureus 43300 0.25 MRSA Defensin2114 Ceftriaxone Staphylococcus aureus 43300 0.25 MRSA Defensin2114 Ceftriaxone Streptococcus pyogenes 12344 0.5
[0109]The results in Table 2 indicate synergy between Defensin2114 and ceftriaxone or penicillin against representative strains of Enterococci, Staphylococci and Streptococci. The results also show synergy between Defensin2114 and penicillin/ceftriaxzone against a methicillin resistant Staphylococcus aureus isolate.
Example 4
Antibiotic Synergism IV
[0110]Synergy between Plectasin and a variety of clinically employed antibiotics was investigated against Staphylococcus aureus ATCC29213, using the procedure described in Examples 2 and 3.
[0111]The antibiotics tested in combination with Plectasin were: Erythromycin, Penicillin G, Gentamicin, Vancomycin, Ciprofloxacin and Cefotaxime. Penicillin G and Cefotaxime are beta-lactam antibiotics. The calculated FIC indices are shown in Table 3.
TABLE-US-00005 TABLE 3 FIC First antibiotic Second antibiotic index Plectasin Erythromycin 1.5 Plectasin Penicillin G 0.5 Plectasin Gentamicin 1.5 Plectasin Cefotaxime 0.5 Plectasin Vancomycin 2 Plectasin Ciprofloxacin 0.75
[0112]Synergism between Plectasin and Penicillin G or Cefotaxime (FIC=0.5) was found against Staphylococcus aureus.
[0113]No FIC antagonism was found between Plectasin and Erythromycin, Gentamicin, Vancomycin or Ciprofloxacin.
Example 5
Using the HMM Files from the PFAM Database to Identify a Defensin
[0114]Sequence analysis using hidden markov model profiles (HMM profiles) may be carried out either online on the Internet or locally on a computer using the well-known HMMER freely available software package. The current version is HMMER 2.3.2 from October 2003.
[0115]The HMM profiles may be obtained from the well-known PFAM database. The current version is PFAM 16.0 from November 2004. Both HMMER and PFAM are available for all computer platforms from, e.g., Washington University in St. Louis (USA), School of Medicine (http://pfam.wustl.edu and http://hmmer.wustl.edu).
[0116]If a query amino acid sequence, or a fragment thereof, belongs to one of the following five PFAM families, the amino acid sequence is a defensin according to the present invention:
[0117]Defensin_beta or "Beta Defensin", accession number: PF00711;
[0118]Defensin_propep or "Defensin propeptide", accession number: PF00879;
[0119]Defensin 1 or "Mammalian defensin", accession number: PF00323;
[0120]Defensin--2 or "Arthropod defensin", accession number: PF01097;
[0121]Gamma-thionin or "Gamma-thionins family", accession number: PF00304.
[0122]An amino acid sequence belongs to a PFAM family, according to the present invention, if it generates an E-value which is greater than 0.1, and a score which is larger or equal to zero, when the PFAM database is used online, or when the hmmpfam program (from the HMMER software package) is used locally.
[0123]When the sequence analysis is carried out locally using the hmmpfam program, it is necessary to obtain (download) the HMM profiles from the PFAM database. Two profiles exist for each family; xxx_ls.hmm for glocal searches, and xxx_fs.hmm for local searches ("xxx" is the name of the family). That makes a total of ten profiles for the five families mentioned above.
[0124]These ten profiles may be used individually, or joined (appended) into a single profile (using a text editor--the profiles are ASCII files) that could be named, e.g., defensin.hmm. A query amino acid sequence can then be evaluated by using the following command line:
hmmpfam-E 0.1 defensin.hmm sequence_file
[0125]wherein "sequence_file" is a file with the query amino acid sequence in any of the formats recognized by the HMMER software package.
[0126]If the score is larger or equal to zero (0.0), and the E-value is greater than 0.1, the query amino acid sequence is a defensin according to the present invention.
[0127]The PFAM database is further described in Bateman et al., 2004, "The Pfam Protein Families Database", Nucleic Acids Research 32 (Database Issue): D138-D141.
Sequence CWU
2140PRTArtificial sequenceSynthetic construct 1Gly Phe Gly Cys Asn Gly Pro Trp Asn Glu Asp Asp Leu Arg Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35 40240PRTPseudoplectania nigrellamat_peptide(1)..(40) 2Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35 40
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text:
Queensland's environment department says an alumina refinery owned by Rio Tinto has spilt sulphuric acid into a creek in central Queensland.
Rio Tinto notified the Department of Environment and Resource Management that the spill occurred at the Yarwun alumina refinery on Sunday during heavy rain.
DERM spokesman Joe Pappalardo said an unknown amount of the acid was released into Boat Creek when the site's stormwater system overflowed in heavy rain.
Urgent inspections and water sampling has been done at Boat Creek and nearby Port Curtis, at Gladstone.
"Inspections by DERM officers . . . have found no evidence of environmental harm suggesting that the recent rain and high tides in Boat Creek have helped to dilute the acid and flush it through the system relatively quickly," Mr Pappalardo told AAP.
"Investigations are continuing, however at this stage DERM has found no evidence that environmental harm has been caused by the spill."
Investigators are looking at the cause of the spill, how much sulphuric acid was involved and the actions taken by Rio Tinto.
Mr Pappalardo said it looks as though it's been a lucky escape for the environment.
"If it's confirmed that a large amount of sulphuric acid has been released without significant environmental harm, then DERM would consider that very fortunate indeed."
"The risk is unacceptable."
A Rio Tinto spokesman told the ABC a faulty drain valve caused the incident and most of the estimated 3000 litres of spilt acid was contained in areas on-site.

subset: commoncrawl


text:
Gordon Shedden sets the pace in the opening BTCC practice session of the weekend at Knockhill
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Gordon Shedden set the pace in the opening Dunlop MSA British Touring Car Championship free practice session of the weekend at Knockhill.
The Honda driver posted a quickest time of 53.395secs at the wheel of his Civic with glorious sunshine bathing the Scottish circuit, with the home hero beating Tom Chilton to top spot by a tenth of a second.
The second Honda of Matt Neal settled into third place as he edged out title rival Jason Plato while Mat Jackson and Andrew Jordan rounded out the top six - despite the latter feeling under the weather after picking up a bug overnight which did leave his participation in the session in some doubt.
Alex MacDowall was seventh, with Rob Collard, Paul O'Neill and Steven Kane rounding out the top ten.
Tom Boardman was eleventh as he switches to the NGTC engine used by Phil Glew at Silverstone last time out, while debutant Jeff Smith was 14th fastest.
James Nash failed to set a representative lap time after a gearbox issue on his car.


text:
The familiar Disney characters are back in Australia with their latest tour, the Disney On Ice Princesses and Heroes.
You and the kids will be entertained by the host, Tinkerbell, along with Cinderella, Ariel, Aurora, Snow White, Jasmine, Belle, Rapunzel, Tiana and all their heroic princes in this breathtaking ice spectacular.
The tour dates and venues are detailed below:
Adelaide – Adelaide Entertainment Centre 14 – 16 June (Fri – Sun)
Newcastle – Newcastle Entertainment Centre 20 – 23 June (Thurs – Sun)
Brisbane – Brisbane Entertainment Centre 27 – 30 June (Thurs – Sun)
Melbourne – Hisense Arena, Melbourne 4 – 8 July (Thurs – Mon)
Sydney – Allphones Arena, Sydney Olympic Park 10 – 14 July (Wed – Sun)
Perth – Perth Arena 19 – 21 July (Fri – Sun)
The good people at Feld Entertainment will be giving away FOUR A Reserve tickets (any configuration) to any tour city for one lucky winner!
All you have to do is answer this question in 50 words or less: What character/trait do you think should a princess/prince have?
Is that question deep enough for you? Goodluck!
Visit their website and Facebook page for more details.
Australian residents only. Competition ends 5pm, 3 May, Friday AEST.


text:
From Best Free Stuff Guide
Graceland coupons help save visitors money on tour admission, and are available on the official website as printable dollars off discounts. Additional coupons are available inside the Memphis Entertainment Coupon Book or from select tourist guides and brochures. Discounts are also available for groups, seniors, students, military personnel, and AAA members.
Graceland is the former home of deceased musician Elvis Presley, also known as "The King." The attraction is located on Elvis Presley Boulevard in Memphis, Tennessee.[1] Parking is available for visitors on-site, and costs $10 for cars and $15 for oversized vehicles.[2]
Flickr: ianloic
|Offered by
|3734 Elvis Presley Boulevard, Memphis, TN 38116
|Phone number
|Three different tours, gift shop
|Types of coupons
|Printable coupons, coupon book, package deals, group discounts
|AAA discount?
|Members save 10% on the Graceland Platinum Tour and tje Graceland Mansion Tour[3]
|AARP discount?
|No, but seniors 62 and over save over $3 off regular adult admission[3]
|Military discount?
|Active and retired military, with valid military ID, save 10% on the Graceland Mansion Tour and the Graceland Platinum Tour[3]
|Allison Hughes
About Graceland
Graceland is a year round attraction, and offers visitors three tour options. These include the Graceland Elvis Entourage VIP Tour, the Graceland Platinum Tour, and the Graceland Mansion Tour. Ticket prices vary by tour, and are available for adults, seniors 62 and over, youth or students 13-18, students with college ID, and children 7-12. Children under the age of seven are admitted free. Ticket prices are as follows:[3]
- Graceland Mansion Tour: adults ($32), seniors ($28.80), youth/students ($28.80), students w/college ID ($28.80), and children ($14)
- Graceland Platinum Tour: adults ($36), seniors ($32.40), youth/students ($32.40), students w/college ID ($32.40), and children ($17.00)
- Graceland Elvis Entourage VIP Tour: $70 for all ages
Graceland Coupons and Free Offers
- The official website for the Memphis Convention & Visitors Bureau offers package deals for Graceland tickets and area hotel accommodations.[4]
- Coupons for Graceland are available from tourist websites like DestinationCoupons.com. The website offers dollars off discounts (in the form of package deals) for the Graceland Wedding Chapel.[5]
- The Memphis Entertainment Coupon Book is available to purchase through the coupon book's official website, and offers a coupon good for a buy one, get one free admission ticket for the Platinum Tour at Graceland.[6]
- Group discounts are available at Graceland. Guests visiting with 15 or more people receive dollars off tour admission. Also, for every 20 people in a group, one complimentary ticket will be issued.[7]
- The official Graceland website promotes a "Specials and Packages" option. This will take visitors to a page that provides information on discounts, coupons, and package deals offered at various times throughout the year. This includes a printable coupon good for $4 off a Platinum Tour or $4 off a VIP Tour.[8] Each coupon is good for up to six admission tickets.
- Discount admission is available for law enforcement and firefighters. A valid ID or membership card must be provided to receive the discount. The offer is valid for 10 percent off the Graceland Mansion Tour and Graceland Platinum Tour.[3]
- Coupons for Graceland may occasionally be offered inside Memphis area tourist guides and brochures, available inside hotel lobbies or visitor centers.[3]
- The free "Elvis Insiders Online Member" club is available on the official website. Members get connected with other Elvis fans, and receive news, discounts, and access to message boards. In addition, for an annual fee of just $19.99 visitors have the option to join the "Basic Elvis Insiders", which gives them a 10 percent discount on admission to Graceland, purchases made at ShopElvis.com, and more. An "Elvis Insiders PLUS" membership costs $39.99 and also includes a membership pin and certificate, a collection of Elvis photos, and a membership card.[9]
- The free Elvis E-newsletter notifies subscribers of special discount offers, contest information, Graceland news, and more by e-mail.[10]
Visiting Graceland
Also See: Free Memphis Zoo Coupons, Free Tennessee Aquarium Coupons, Free Dollywood Coupons, Free Nashville Zoo Coupons, Free Country Music Hall of Fame Coupons, Free Aquarium Coupons, Free Zoo Coupons, Free Museum Coupons
- ↑ http://www.elvis.com/graceland/maps_and_directions.aspx
- ↑ http://www.elvis.com/graceland/faq.aspx
- ↑ 3.0 3.1 3.2 3.3 3.4 3.5 http://www.elvis.com/graceland/tours/tickets-reservations/default.aspx
- ↑ http://www.memphistravel.com/special-offers
- ↑ http://www.destinationcoupons.com/nevada/las_vegas/Graceland-Wedding-Chapel/wedding-chapel-discounts.asp
- ↑ http://www.entertainment-savings-offers.com/memphis/book/tickets/list/
- ↑ http://www.elvis.com/graceland/groups/group_rates_reservations.aspx
- ↑ http://www.elvis.com/graceland/specials_and_packages.aspx
- ↑ http://www.elvis.com/news/detail.aspx?id=2143
- ↑ http://www.elvis.com/news/follow-elvis/e-newsletter.aspx


text:
Pressure Transducer, Range 0 to 30 psi
ASHCROFT| Zoro #: G0133016| Mfr #: K17MO242C130#XFM
Zoro #: G0133016Mfr. Part #: K17MO242C130#XFM
$398.75 /ea
This item ships free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G0133016 Mfr #: K17MO242C130#XFM
|Includes: Instructions
|Item: Pressure Transmitter
|Range: 0 to 30 psi
|Compatible With: Any Process Compatible with 316Stainless Steel
|Power Required: 9 to 36VDC
|Accuracy: +/-0.5% Full Scale
|Max. Pressure (PSI): 60
|Manufacturers Warranty Length: 1 year
|Loop Resistance: Dependent on Loop Supply Voltage
|Process Connection: 1/4" NPT Male
|Wetted Materials: 316 Stainless Steel
|Output: 4 to 20 mA at 12 to 30VDC
|Operating Temp.: -20 To 180 F
|Housing: Stainless Steel
|Electrical Connections: 1/2" Conduit
|Safety Rating: Class 1, Div. 2, Groups A, B, C, and D
|NEMA Rating: 4X, Non-Incendive
|Lead Length (In.): 36
|Thermal Error: +/-.04% per Degree F from -20 to 168F
|Country of Origin (subject to change): United States
Product Description
Pressure Transducer, Range 0 to 30 psi, Accuracy +/-0.5% Full Scale, Output 4 to 20 mA at 12 to 30VDC, Power Required 9 to 36VDC, Process Connection 1/4 In NPT Male, Electrical Connections 1/2 In Conduit, Lead Length 36 In, Wetted Materials 316 Stainless Steel, Housing Stainless Steel, Operating Temp -20 To 180 F, NEMA Rating 4X, Non-Incendive, Safety Rating Class 1, Div. 2, Groups A, B, C, and D, Max Pressure 60 PSI, Includes Instructions


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This Echo chainsaw features a computer-controlled engine ignition timing system to help ensure cutting efficiency. The 45 cc engine is designed to provide ample power during use, and the saw uses an automotive-style air filter and an engine air pre-cleaner to help extend the engine's lifespan.
- 20 in. bar and 20 in. chain are suitable for a variety of commercial and residential applications
- Professional-grade 2-stroke, 45cc engine for powerful performance
- Digital ignition is designed to automatically adjust engine timing for cutting efficiency
- Automotive style air filter and engine air pre-cleaner to help ensure a long engine life
- Certified with the highest EPA engine durability rating
- Inertia-type safety chain brake to help ensure safety
- Automatic, adjustable, clutch-driven oiler for convenience
- Includes operator's manual and engine oil for ease of use


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I'm a bit fond of the forthcoming Autumn 2010 Levi's Vintage (I still prefer LVC) collection – especially the rework on the denim jacket. The 1936 Type 1 looks to be a strong look. It is almost wearable as a heavy shirt based on the fit of this model. "Heavy weight denim jacket in a vintage 'Graniteville' finish – a heavily distressed look with colouration to the denim and a crumpled finish to the fabric. Rivets are exposed, sleeve seams are worn and there's a back cinch for detail." Pre-order at Oki-ni.
More looks at the after the click.


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Welcome to L A Hauer-LaDuca Architect P.C.
We would like to take this opportunity to thank you for looking at our site,
we encourage you to look at our projects both current and completed.
You may do this by clicking on the projects tab to the right, a sub-menu will bring up the different building types that we have done.
Please re-visit often, as construction continues!!
Our Architectural Team handles both Residential and Commercial Design with more than 25 years of experience.
We have experience with the following building types.
We are NCARB certified, and currently Licensed in New York State.
2015 Buffalo Brick by Brick Finalist for Best New Construction
and Best Multi- Residential Project.
Supportive Housing Project of the year 2016'- Recognized by The Supportive Housing Network of New York.
Hope Garden
Apartments for Homeless Women
Buffalo, New York.
Sponsered by the Matt Urban Center.
Current Featured Project:
Cornerstone CFCU
4255 Witmer Road Niagara, New York
St. Bridget's Church & St. Brendan's Parish Sanctuary Reconstruction Newfane, New York.
Current Projects:
See also "Projects Tab"
Cornerstone CFCU
New Community Federal Credit Union Branch
Town of Niagara, New York.
St. Bridget's Church & St. Brendan Parish
Sanctuary Remodel
Newfane, New York.
William George Agency for Children Services
Twenty-four bed Cottage.
Dryden, New York.
Holy Redeemer Cemetery Mausoleum.
Albany, New York.
Mt. Hebron Memorial Park Mausoleum.
Montclaire, New Jersey.
See also "Projects Tab"
Empire One Community Federal Credit Union
Branch & Corporate Offices.
Orchard park, New York.
Grand Opening Scheduled for September 2016
Evans Bank
Branch Reconstruction.
Lancaster, New York.
Hope Garden
Apartments for Homeless Women
Buffalo, New York.
Sponsered by the Matt Urban Center.
2015 Buffalo Brick by Brick Finalist for Best New Construction & Best Multi- Residential Project.
Supportive Housing Project of 2016- Recognized by The Supportive
Housing Network of New York.
Catholic Center Annex Building- Walsh Insurance Agency
Facade modifcation,
Buffalo, New York.
St. Martha's Parish
Altar Modifications and Interior finishes.
Depew, New York.
Myrtle Hill Mausoleum
Myrtle Hill Cemetery
Rome, Georgia.
St. Anthony Mausoleum
Addition to Holy Sepulchre Cemetery Mausolueum
Cheektowaga, New York.
Cassetta Insurance Agency
Facade Modification & Office Planning
Buffalo, New york.
George Junior Republic, UFSD
Boscowitz, Gym & Pool Reconstruction
Freeville, New York.
Evans Bank Branch & Insurance Agency Office Lockport, New York.
George Junior Republic, UFSD
Education Unit #1, Classroom Addition,
Freeville, New York.
George Junior Republic, UFSD
Ewald Dining Facility Reconstruction
Freeville, New York
Nativity of the Blessed Virgin Mary
School Office Suite
Williamsville New York.
Corpus Christi Parish Rectory
Vestibule Reconstruction.
Buffalo, New York.
Response To Love Center
New Elevator
Buffalo, New York.


text:
Android on PCs, that's definitely been thought of and done before, if the number of Android-powered all-in-one's and laptops of late is any indication. However, Mobile Media Ventures, the people behind iConsole.tv have an even more ambitious goal: to run Android side by side Windows on any PC of your choice.
Perhaps iConsole.tv's Unit 00 unveiled last year will speak to the company's chops. Or perhaps the relatively unknown brand will make others wary. Whatever the case may be, Console OS has a dream for Android on the PC, and that dream is quite enormous. Android has had various ports for Intel (or AMD) hardware for a long time now, ranging from Android-x86 to emulators to the versions shipped by OEM's like ASUS or Acer. None, however, come close to the matrix of features that Console OS aims to deliver. First of all, you've got an easy dual-boot option, or at least simply dual-boot in the beginning, with the easy part coming later down the road. You've got complete Android 4.4 compatibility, versus the buggy or partial versions shipped by other similar endeavors. And perhaps most interestingly, you've got Google Play Store support, or so the team claims.
Console OS is being marketed not only towards casual or even every day users of Android but especially to gamers also. This subset of Android users have the most to benefit with hardware that go beyond the limited power and capabilities of even the mightiest tablet or smartphone. As far as hardware is concerned, Console OS is promised to work on many PCs, but the number of supported models are actually not exhaustive. Many are laptops but quite a few can be identified as tablets or hybrids. Console OS claims that this is because, like Android on mobile devices, Console OS has to build Android for specific devices as well to ensure full compatibility. Other makes and models can be added, but that depends on the votes of Kickstarter backers.
Yes, this is a Kickstarter thing, and this is probably where the dream starts to fall apart for some. This isn't a finished product yet, though Console OS already has a head start, at least as far as basic functionality goes. There are still some missing bits, some of them quite important. InstaSwitch is one, which will allow users to switch between Wndows and Android at the push of a button. WindowFlinger is another, which brings true windows (with the lowercase "w") to Android, allowing things like running Android apps side by side. Perhaps most worrying for existing Android users is that there technically isn't any Google Play Store, but Console OS promises that they will be able to import Play Store purchases easily
Console OS also wants to target OEMs, and though they might find interested ears and supportive voices there, they will most likely also find the biggest resistance, if the stillborn Samsung ATIV Q and ASUS Transformer Book Duet are any indication. Still, if you believe in the vision and trust in the makers, this Kickstarter might be the best time for you to jump in. At $10, which is a limited slot offer, you can get one copy of Console OS, with free upgrades for life. After Kickstarter, it will cost $19.99 a year. Console OS plans to have a developer release around September and a 1.0 release by December.
SOURCE: Kickstarter


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Eric Decker returned a punt 90 yards for a touchdown to help Denver get within 16-13 of Oakland through three quarters Monday night in a game that featured Sebastian Janikowski's NFL record-tying 63-yard field goal.
Matt Prater hit a 30-yard field goal late in the third when a promising Broncos drive stalled out near the goal line.
Just seconds before halftime, Janikowski kicked the ball low and watched it barely creep over the crossbar. Janikowski shares the record with Jason Elam and Tom Dempsey.
His teammates ran onto the field to celebrate before heading into the locker room.
It was one of three field goals Janikowski had in the second quarter, the other two being much shorter. Fullback Marcel Reece also hauled in a TD pass.
The Broncos climbed back into the game early in the third quarter thanks to their special teams. Decker fielded Shane Lechler's 57-yard punt and was off to the races.
Upon reaching the end zone, Decker headed for the fans and jumped into the stands, his rendition of the "Lambeau Leap."
The Broncos later went on a 12-play drive that culminated with Prater's kick.
It was a first half featuring big plays for the Raiders, who capitalized on a blocked punt and a Broncos fumble.
Just before the end of the first quarter, Darryl Blackstock broke through the line and got a hand on Britton Colquitt's punt, setting Oakland up with excellent field position at the Denver 42.
Jason Campbell found a wide open Reece for a 3-yard score.
On Denver's next offensive play, Knowshon Moreno fumbled the ball away after a hit by Quentin Groves.
As the Raiders were celebrating, the rain began to steadily fall. Oakland couldn't move the ball in the downpour and settled for a 37-yard field goal by Janikowski.
Soon after that, the rain stopped.
The Broncos were driving late in the second quarter but couldn't convert on a long third down. They elected to have Prater try a 56-yard field goal, which sailed wide right and gave the Raiders great field position.
Campbell moved the Raiders down the field and Janikowski hit a 21-yarder with 1:27 remaining until halftime.
With plenty of time left before intermission, Kyle Orton had the Broncos on the move but was picked off by Matt Giordano at the Oakland 24.
A 15-yard facemask penalty on Broncos defensive tackle Kevin Vickerson put the ball near midfield and Campbell's throw to Reece moved the Raiders to the Denver 45 and put Janikowski within range of history.
Dempsey set the record for New Orleans on Nov. 8, 1970, and Denver's Elam matched it on Oct. 25, 1998.
Janikowski connected on a 61-yard field goal on Dec. 27, 2009.
Oakland fumbled on its opening possession of the season when rookie linebacker Von Miller knocked the ball loose with his helmet after a catch by Jacoby Ford. Miller was the No. 2 overall pick by the Broncos.
Fellow rookie Rahim Moore pounced on the loose ball. The Broncos couldn't capitalize as Orton completed just one short pass on three attempts, leading to Prater's 28-yard field goal.
The much-maligned Denver defense did a formidable job early on as the unit also forced the Raiders to punt.
However, Elvis Dumervil walked off the field holding his shoulder, throwing his helmet down in disgust. He later returned to the field but played sparingly.
Dumervil, who led the league in sacks in 2009, missed all of last season with a torn pectoral muscle.
This was a game featuring two new head coaches. Hue Jackson took over in Oakland after serving as offensive coordinator last season, while John Fox was in his first game in charge of the Broncos after arriving from Carolina.
The Broncos are trying to get their franchise back on track a year after turning in a franchise-worst 4-12 season. They brought in Fox and turned the reins over to Hall of Famer John Elway, who's serving as chief of football operations. The organization even renamed their stadium (Colorado-based Sports Authority recently took over the naming rights from Invesco and completed its sign out front just in time for the opener).
Still, it's difficult to erase all the memories from an abysmal 2010.
One of the low points for the Broncos in a season full of worthy candidates was an embarrassing 59-14 thumping by the Raiders on Oct. 24. It could've been even worse but the Raiders relented in the fourth quarter, showing mercy toward one of their most bitter rivals.
Darren McFadden led the way, shredding the porous Denver defense for 165 yards rushing and three scores.
On Monday, facing an overhauled Denver defensive front, McFadden had another solid game going, rushing for 100 yards on 19 carries through three quarters.


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Cherry Cinnamon Cobbler Recipe
My mom made this recipe when I was very young. Its cheery color and cinnamon scent make it just right for gray, winter days.
- 1 can (16 ounces) pitted tart red cherries
- 1/2 cup sugar
- 2 to 4 tablespoons red cinnamon candies
- 2 tablespoons cornstarch
- 1/2 cup water
- 1-1/2 cup all-purpose flour
- 2 teaspoons baking powder
- 1/2 teaspoon salt
- 6 tablespoons brown sugar, divided
- 1/3 cup chopped nuts
- 1/4 cup shortening
- 1 large egg, beaten
- 2 tablespoons whole milk
- 1 tablespoon butter
- 1/4 teaspoon ground cinnamon
- 1. Drain cherries, reserving juice. In a saucepan, combine sugar, candies and cornstarch. Stir in water and juice. Cook over medium heat, stirring occasionally, until thickened. Stir in cherries. Pour into an 8-in. square baking pan; set aside.
- 2. In a bowl, combine flour, baking powder, salt, 3 tablespoons brown sugar and nuts. Cut in shortening until crumbly. Add egg and milk; mix until dry ingredients are moistened (add a few drops more milk if necessary).
- 3. Roll out dough; brush with butter. Combine cinnamon and remaining brown sugar; sprinkle over dough. Roll up; cut into 16 slices. Lay slices over cherry filling. Bake at 400° for 35-30 minutes. Serve warm. Yield: 8 servings.
Nutritional Facts
1 piece: 347 calories, 12g fat (3g saturated fat), 31mg cholesterol, 134mg sodium, 57g carbohydrate (36g sugars, 1g fiber), 5g protein.
Reviews for Cherry Cinnamon Cobbler
Average Rating
Reviewed Feb. 15, 2016
"I made this recipe over 55 years ago. I found the recipe in a book that myMom had pasted recipes she cut out of magazines. I was my first time I made a recipe that I chose the recipe and made myself. It is very good."
© 2017 RDA Enthusiast Brands, LLC


text: Just a few seasons ago they had a team of Arenas, Hughes, Jamison and several other very talented players, but what did they do, they traded away threee future allstars for very little if anything. And some of you think the Pacers have made a few made trades over the years. Once again we can learn from this, NEVER TRADE YOUNG TALENTED PLAYERS TOO EARLY.


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While Rihanna's been flaunting her figure in an array of revealing swimwear while vacationing in Hawaii this week, another star opted for more modest beach attire.
Florence and the Machine frontwoman Florence Welch was spotted on the beaches of Rio De Janeiro, Brazil in a high-waisted black bikini and a red swimming cap Tuesday.
And the "Shake it Off" singer, 25, isn't the only celeb who seems to prefer a covered-up look.
Ke$ha, 24, who's known for her daring style, also wore a high-waisted two-piece at an Australian beach in March 2011, while Taylor Swift, 22, rocked a modest polka dot number in the Bahamas back in June 2010.
Tell Us: Would you wear a retro bikini or bathing suit?


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Who is Selling You Chiquilitros of Gasoline? Evidence From a Public Disclosure Policy
This paper estimates the impacts of disclosing information online and through the newspapers of gas stations that violate fuel supplying standards in Mexico. Using data from inspection histories, it finds that disclosing information online decreases the probability that any gas station would be found in violation in subsequent periods. Gas stations exposed in the newspapers are estimated to decrease their sales at the month of the newspaper publication. This effect fades with time and is not significant for subsequent months. The paper shows that public disclosure mechanisms can complement enforcement effort in contexts where institutions are weak.
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|Date of creation:
|May 2012
|Contact details of provider:
| Web page: http://www.banxico.org.mx
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References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Konar, Shameek & Cohen, Mark A., 1997. "Information As Regulation: The Effect of Community Right to Know Laws on Toxic Emissions," Journal of Environmental Economics and Management, Elsevier, vol. 32(1), pages 109-124, January.
- Stefano DellaVigna & Matthew Gentzkow, 2010.
"Persuasion: Empirical Evidence,"
Annual Review of Economics,
Annual Reviews, vol. 2(1), pages 643-669, 09.
- Stefano DellaVigna & Matthew Gentzkow, 2009. "Persuasion: Empirical Evidence," NBER Working Papers 15298, National Bureau of Economic Research, Inc.
- Beach, Robert H. & Kuchler, Fred & Leibtag, Ephraim S. & Zhen, Chen, 2008. "The Effects of Avian Influenza News on Consumer Purchasing Behavior: A Case Study of Italian Consumers' Retail Purchases," Economic Research Report 56477, United States Department of Agriculture, Economic Research Service.
- Gary S. Becker, 1974. "Crime and Punishment: An Economic Approach," NBER Chapters,in: Essays in the Economics of Crime and Punishment, pages 1-54 National Bureau of Economic Research, Inc.
- Gary S. Becker, 1968. "Crime and Punishment: An Economic Approach," Journal of Political Economy, University of Chicago Press, vol. 76, pages 169-169.
- García, Jorge H. & Sterner, Thomas & Afsah, Shakeb, 2007. "Public disclosure of industrial pollution: the PROPER approach for Indonesia?," Environment and Development Economics, Cambridge University Press, vol. 12(06), pages 739-756, December.
- Afsah, Shakeb & Sterner, Thomas & López, Jorge, 2004. "Public Disclosure of Industrial Pollution: The PROPER Approach for Indonesia?," Discussion Papers dp-04-34, Resources For the Future.
- García López, Jorge & Sterner, Thomas & Afsah, Shakeb, 2009. "Public Disclosure of Industrial Pollution: The PROPER Approach for Indonesia?," Working Papers in Economics 414, University of Gothenburg, Department of Economics.
- Wesley A. Magat & W. Kip Viscusi, 1992. "Informational Approaches to Regulation," MIT Press Books, The MIT Press, edition 1, volume 1, number 026213277x, July.
- Claudio Ferraz & Frederico Finan, 2008. "Exposing Corrupt Politicians: The Effects of Brazil's Publicly Released Audits on Electoral Outcomes," The Quarterly Journal of Economics, Oxford University Press, vol. 123(2), pages 703-745.
- Ferraz, Claudio & Finan, Frederico S., 2007. "Exposing Corrupt Politicians: The Effects of Brazil's Publicly Released Audits on Electoral Outcomes," IZA Discussion Papers 2836, Institute for the Study of Labor (IZA).
- Hamilton, James T, 1999. "Exercising Property Rights to Pollute: Do Cancer Risks and Politics Affect Plant Emission Reductions?," Journal of Risk and Uncertainty, Springer, vol. 18(2), pages 105-124, August.
- Khanna, Madhu & Quimio, Wilma Rose H. & Bojilova, Dora, 1998. "Toxics Release Information: A Policy Tool for Environmental Protection," Journal of Environmental Economics and Management, Elsevier, vol. 36(3), pages 243-266, November.
- Ginger Zhe Jin & Phillip Leslie, 2003. "The Effect of Information on Product Quality: Evidence from Restaurant Hygiene Grade Cards," The Quarterly Journal of Economics, Oxford University Press, vol. 118(2), pages 409-451.
- Graff Zivin, Joshua & Neidell, Matthew, 2009. "Days of haze: Environmental information disclosure and intertemporal avoidance behavior," Journal of Environmental Economics and Management, Elsevier, vol. 58(2), pages 119-128, September.
- Joshua Graff Zivin & Matthew Neidell, 2008. "Days of Haze: Environmental Information Disclosure and Intertemporal Avoidance Behavior," NBER Working Papers 14271, National Bureau of Economic Research, Inc.
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text:
Eliza E. Rathbone, William H. Robinson, Elizabeth Steele, and Marcia Steele With contributions by Sylvia Albro, H. Travers Newton and Galina K. Olmsted
Popular perceptions of Vincent van Gogh (1853–1890) frequently envision the painter working before nature in an intense frenzy, yet the artist's method was often controlled and deliberate. Van Gogh Repetitions is the first book to focus on the artist's "repetitions," a term he used to describe his process of producing more than one version of a composition. In this groundbreaking and beautifully illustrated book, a series of essays considers the many unresolved issues and controversies surrounding the repetitions, including their origins, development, and meaning in van Gogh's art. Technical and analytical examinations provide new insights into the artist's working methods and approach to the creative process. Catalog to the exhibition, Van Gogh Repetitions ( March 2, 2014 to May 26, 2014)
125 Color Illustrations
200 pages
9.5" x 10"
Published 2013
Original price: $50.00
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text:
Ohio Lawmakers Consider Bill To Privatize Job Creation
Ohio lawmakers are considering a bill that would create a private board to create jobs and entice businesses to locate in Ohio. The Ohio Department of Development, the state agency now in charge of job development in the buckeye state, would still exist but the private board, which would be operated by private business leaders, would be able to move at what Governor Kasich calls "the speed of business."
By reducing regulation and meeting the needs of businesses faster, Kasich and the republicans who control all of state government at this point, hope to invigorate Ohio's economy. In the mid 90's, then Florida Governor Lawton Chiles created Enterprise Florida. Though not identical, that quasi private economic development board was to do many of the same things JobsOhio would be charged with doing. Now, newly elected Florida Governor, Republican Rick Scott, has decided to fire the head of that board.
Scott will create a new Florida Commerce Department and will put Enterprise Florida under it. James Call is the Legislative News Producer for Florida Public Radio. In an interview with Ohio Public Radio's Jo Ingles, Call explains what that state's board was supposed to do and why it is now being changed somewhat.
Call says this change should lead to more public accountability. Call says Floridians hungry for jobs right now. The unemployment rate in the Sunshine State hovers around 12 percent….at least two points higher than Ohio's. Call says Florida is hoping the changes will attract at least one fortune 500 company to someday locate in that state.


text:
Motorola - (RMN5126A)Item#: 9114809 | Model#: RMN5126A
- Price:
- $2,879.99
- Shipping:
- Temporarily out of stock. Order today and we'll deliver when available. (Details)
Motorola - Product Details
Manufactured by: Motorola
Warranty provided by: Motorola
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Mfg Part No: RMN5126A
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( Length:0, Width:0, Depth:0)
Shipping Weight: 0.1000 pound(s)
Click here for full warranty and support information
- Motorola logos are registered trademarks of Motorola. All others trademarks and copyrights mentioned herein are the property of their respective owners.


text:
You Are Here: AHRQ Archive Home > Emergency Preparedness Resource Inventory (EPRI) > Technical Manual > Chapter 1
This information is for reference purposes only. It was current when produced and may now be outdated. Archive material is no longer maintained, and some links may not work. Persons with disabilities having difficulty accessing this information should contact us at: https://info.ahrq.gov. Let us know the nature of the problem, the Web address of what you want, and your contact information.
Please go to www.ahrq.gov for current information.
With funding from the Agency for Healthcare Research and Quality (AHRQ), Abt Associates and Geisinger Medical Systems, together with the Web development firm RelyonMedia, created and pilot tested (in an eight-county area of eastern Pennsylvania) a Web-based tool named Emergency Preparedness Resource Inventory—A Tool for Local, Regional, and State Planners (EPRI) for compiling an inventory of healthcare and other emergency resources. Organizations that control these resources can log on to EPRI and enter and update information on their resources. These organizations, as well as emergency response planners and other government officials, can also run a variety of reports from the inventory that provide detailed and summary information on available resources. System administrators can customize EPRI—for example, by specifying what resources are included in the system and what specific reports users can (and cannot) run.
To use EPRI, jurisdictions must download it from the AHRQ Web Site and then install it on a Web server. That is, jurisdictions cannot go to an existing Web Site and start using EPRI immediately.
This manual is a technical description of EPRI and is intended primarily for system administrators charged with installing, customizing, and hosting EPRI. Following this introductory section, the manual contains six sections and an appendix:
In addition to this technical manual, the project produced two other reports:
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text:
The Seymour Duncan Antiquity Humbuckers are made to look and sound like a well-worn '50s humbucker as originally designed by Seth E. Lover. The pickups are built to traditional standards. Each has its... Click To Read More About This Product
The Seymour Duncan Antiquity Humbuckers are made to look and sound like a well-worn '50s humbucker as originally designed by Seth E. Lover. The pickups are built to traditional standards. Each has its own character and DC resistance, just like the originals. The nickel silver cover and the poles are aged to give the player a pickup that looks like it's been played regularly for 40 years.
The neck position model includes a front-tapered mounting ring and the screws that go with it.
Like the neck position model, the bridge position humbucker is a faithful replica of the great sounding "patent applied for" humbuckers that Seth E. Lover designed in 1955. The bridge pickup is wound a little hotter and better balanced for its position. The mounting ring is slightly higher for the increased pickup height.
Both pickups are not wax potted to retain the sound and appearance of the originals. Again, each pickup is built by hand in the "Antiquity" room and every magnet and bottom plate are signed to guarantee that you are getting an original Seymour W. Duncan Antiquity. Hookup wire includes the classic braided "push back" cloth braid wire.
To some, this is craziness, intentionally creating an aged electrical component. But if you're like Seymour Duncan, and you appreciate the personal commitment and hand craftsmanship found in early electric guitars, then you'll want to capture that timeless beauty with an Antiquity pickup.
Reviewed by 3 customers
Comments about Seymour Duncan Antiquity Humbucker Pickup:
I had the Gibson 490R and 498T pair in my Gibson SG Standard and never really cared for them. I found them to be muddy and not clear enough for my ears. I threw these bad boys in as a reccomendation and they breathed new life into my guitar. It sounds fantastic and vintage sounding. This comes as close to a PAF as I have ever heard especially when compared to my friends vintage Gibson. The clarity is amazing and is very friendly to overdrive. I play my SG through various effects into a 65 Super Reverb Re-issue and play mostly classic rock, blues, and funk and it fits my needs. I highly reccomend.
Comments about Seymour Duncan Antiquity Humbucker Pickup:
Simply put, his pickups just sound great. I bought this one to replace a Gibson 57 Classic Plus that was a little too bright for a project Strat I've been tweaking. (Nothing against the 57. My Poplar body/Pau Ferro board needed some warming up.) The Antiquity was just the ticket, sweet where the '57 was a bit harsh, warmer the '57 was a bit too bright. This pickup is rich without being wooly, with just the right amount of sizzle. And the harmonics just fly off this thing. Very nuanced tone, but nice and gritty too. If your rock travels take you from Moby Dick to Eruption and back down to La Grange you will not be disappointed. I've got everything from Joe Bardens to Wolfetones to Bill Lawrence stuff wound by the man himself. Love 'em all, but I love these Seymour Duncans just as much. Reasonably priced too. Tag even comes with hand-written output measurement, so you know your pickup was tested and is unique, as all hand-wound pups are. Highly recommended.
Best Uses
Comments about Seymour Duncan Antiquity Humbucker Pickup:
Replaced the 490R pup in the neck position of a Gibson ES-775 (an upscale ES-175), 16 inch maple body and neck. Was looking for the ES-175 early PAF sound and found it, even after replacing the nickel cover and pole pieces with the 490's gold-plated parts. The 490 was fine in small venues at modest volume levels. The Antiquity seems to provide more depth, clarity and responsiveness within a broader range of performing situations. There are also more, and more easily accessible tones now when using both the neck and bridge pickup simultaneously.
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