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Patent application title: Antibodies immunoreactive with mutant hydroxypenylpyruvatedioxygenase
Vaithilingam Sekar (Ames, IA, US)
Vaithilingam Sekar (Ames, IA, US)
Bruce Held (Ames, IA, US)
Bruce Held (Ames, IA, US)
Kyu Chung (Basil, CH)
Paul F. Russell, Jr. (Portage, MI, US)
IPC8 Class: AC12P2100FI
USPC Class:
435 696
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition recombinant dna technique included in method of making a protein or polypeptide blood proteins
Publication date: 2011-05-05
Patent application number: 20110104755
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Patent application title: Antibodies immunoreactive with mutant hydroxypenylpyruvatedioxygenase
Vaithilingam Sekar
Bruce Held
Paul F. Russell, JR.
Kyu Chung
Origin: ,
IPC8 Class: AC12P2100FI
USPC Class:
Publication date: 05/05/2011
Patent application number: 20110104755
Antibodies immunoreactive to mutant Pseudomonas HPPD are provided, and in
an embodiment the mutant HPPD is one in which the wild-type HPPD is
substituted at residue 336 with tryptophan for glycine. Also provided are
hybridomas producing the antibodies, as well as methods of making and
using the antibodies.
1. An antibody immunoreactive with a Pseudomonas HPPD polypeptide,
wherein said antibody is selected from the group consisting of: (a) an
antibody immunoreactive with a mutated HPPD polypeptide wherein said
mutated HPPD polypeptide comprises a mutation substituting tryptophan at
residue 336 when compared to a wild-type HPPD polypeptide; (b) an
antibody immunoreactive with a mutated HPPD polypeptide wherein said
mutated HPPD polypeptide comprises a mutation substituting tryptophan at
residue 336 when compared to a SEQ ID NO: 4; (c) an antibody
immunoreactive with a polypeptide comprising the polypeptide encoded by
SEQ ID NO: 2; (d) an antibody immunoreactive with a polypeptide encoded
by SEQ ID NO: 3; (e) an antibody produced by a hybridoma selected from
the group consisting of 1C5 assigned ATCC Number PTA-10312, 2E6 assigned
ATCC number PTA-10313, and 6H11 assigned ATCC number PTA-10314; and (g) a
fragment of an antibody of (a), (b), (c), (d), (e) or (f) wherein said
fragment is immunoreactive with a mutated HPPD polypeptide selected from
the group consisting of the polypeptides of (a), (b), (c), and (d).
2. The antibody of claim 1, wherein said antibody is immunoreactive with the polypeptide of SEQ ID NO: 2.
3. The antibody of claim 1, wherein said antibody is immunoreactive with the polypeptide encoded by SEQ ID NO: 3.
4. The antibody of claim 1, wherein said antibody is immunoreactive with a mutated HPPD polypeptide wherein said mutated HPPD polypeptide comprises a mutation substituting tryptophan at residue 336 when compared to a wild-type HPPD polypeptide.
5. The antibody of claim 1, wherein said wild-type HPPD polypeptide is SEQ ID NO: 4.
6. The antibody of claim 1, wherein said antibody is not immunoreactive with a soybean wild-type HPPD polypeptide.
7. The antibody of claim 1, wherein said antibody is a polyclonal antibody.
8. An antibody produced by a hybridoma selected from the group consisting of 1C5 assigned ATCC Number PTA-10312, 2E6 assigned ATCC number PTA-10313, and 6H11 assigned ATCC number PTA-10314
9. A hybridoma cell line selected from the group consisting of 1C5 assigned ATCC Number PTA-10312, 2E6 assigned ATCC number PTA-10313, and 6H11 assigned ATCC number PTA-10314.
10. A method for detecting the presence of a mutated Pseudomonas HPPD polypeptide in a composition, the method comprising contacting said composition with the antibody of claim 1, and determining whether the antibody is immunoreactive with any of said mutated HPPD polypeptide in said composition.
111. The method of claim 10, wherein said antibody is not immunoreactive with a wild-type HPPD polypeptide.
12. The method of claim 10, wherein said antibody is a polyclonal antibody.
13. A method of generating an antibody, the method comprising (a) immunizing an animal with a Pseudomonas polypeptide selected from the group consisting of (i) a mutated HPPD polypeptide wherein said mutated HPPD polypeptide comprises a mutation substituting tryptophan at residue 336 when compared to a wild-type HPPD polypeptide; (ii) a mutated HPPD polypeptide wherein said mutated HPPD polypeptide comprises a mutation substituting tryptophan at residue 336 when compared to a SEQ ID NO: 4; (iii) a polypeptide comprising the polypeptide encoded by SEQ ID NO: 2; (iv) a polypeptide encoded by SEQ ID NO: 3; (v) a fragment of a polypeptide of (i), (ii), (iii) or (iv) wherein said fragment generates an antibody in said animal immunoreactive to a polypeptide selected from the group consisting of (i), (ii), (iii) or (iv); (b) recovering splenocytes from the immunized animal; (c) fusing said splenocytes with myleoma cells to produce monoclonal hybridomas; and (d) producing monoclonal antibodies from said hybridoma.
14. A kit for detecting the presence of a mutated Pseudomonas HPPD polypeptide in a composition, the kit comprising the antibody of claim 1, and a detection agent.
16. The kit of claim 13, wherein said kit further comprises (i) a plant sample obtaining device; (ii) a support having affixed thereto said antibody which is capable of forming a binary complex with said mutated HPPD polypeptide which may be present in said sample; and a binary complex detecting means.
17. A kit for detecting the presence of a HPPD polypeptide in a composition, the kit comprising an antibody produced by a hybridoma selected from the group consisting of 1C5 assigned ATCC Number PTA-10312, 2E6 assigned ATCC number PTA-10313, and 6H11 assigned ATCC number PTA-10314.
[0001] The hydroxyphenylpyruvate dioxygenases are enzymes which catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. This reaction takes place in the presence of iron and in the presence of oxygen (Crouch, N. P. et al., Tetrahedron, 53, 20, 6993-7010, 1997). It may be hypothesized that the HPPDs contain an active site which is capable of catalyzing this reaction, in which iron, the substrate and the molecule of oxygen link together.
[0002] Some molecules which inhibit this enzyme, and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate, are also known. Some of these molecules have been used as herbicides since inhibition of the reaction in plants leads to whitening of the leaves of the treated plants and to the death of the said plants (Pallett, K. E. et al. 1997 Pestic. Sci. 50 83-84). The herbicides for which HPPD is the target, and which are described in the state of the art, are, in particular, isoxazoles (EP418175, EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, which is a selective herbicide for maize, diketonitriles (EP496630, EP496631), in particular 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2-phenyl)propane-1,3-dione, triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particular sulcotrione, or else pyrazolinates.
[0003] In addition, the amino-acid sequence of hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 has been described (Ruetschi et al.: Eur. J. Biochem. 205, 459-466, 1992). At U.S. Pat. No. 6,268,549, a sequence of a gene of this type is described and that such a gene can, once incorporated into plant cells, produce an over-expression or an activation of HPPD in the plants giving to the latter an worthwhile tolerance to certain novel herbicides, such as those of the isoxazoles family or that of the triketones. The sequence can be of bacterial origin, such as especially the genus Pseudomonas or alternatively of plant origin, such as especially of monocotyledonous or dicotyledonous plants, especially of Arabidopsis or of Umbelliferae, such as, for example, the carrot (Daucus carotta). It can be isolated native or wild or possibly mutated artificially while at the same time fundamentally retaining a property of herbicidal tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family or that of the triketones.
[0004] Also described at U.S. Pat. No. 6,245,968 is a mutant of such HPPD sequences. It is useful in the process of over expressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant sufficient to have enough functional enzyme in spite of the presence of the inhibitor. The patent describes that by mutating the enzyme in the vicinity of its C-terminal part, it was possible to obtain functional (enzymatically active) HPPDs which were less sensitive to HPPD inhibitors, such that expression of these functional HPPDs in plants improves the tolerance of the plants to HPPD inhibitors. Mutants which are enzymatically active or functional retain a significant portion of HPPD catalytic activity, and in the case of plants transformed with the sequences, the mutated sequences should preferably retain sufficient HPPD activity to sustain the growth of the plant.
[0005] With this mutation an amino acid of the primary sequence is replaced with another amino acid. By aligning these known sequences, by using the customary means of the art, such as, for example, the method described by Thompson, J. D. et al, (CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22; 4673-4680, 1994), and accessing these computer programs for sequence alignment which include a wide variety of tools, and are accessible via the Internet, for example, the skilled person is able to define the sequence homologies in relation to a reference sequence and find the key amino acids or else define common regions, making it possible, for example to align the sequence and locate a referenced position and to define a C-terminal region and an N-terminal region on the basis of this reference sequence.
[0006] In the case of the present invention, the reference sequence is the Pseudomonas sequence, with all the definitions and indications of the positions of particular amino acids being made with respect to the primary Pseudomonas HPPD sequence, here SEQ ID NO: 4 (this is the sequence identified as sequence 31 of the '968 patent). That reference sequence is shown in FIG. 1 of the '968 patent and depicts an alignment of several HPPD sequences which are described in the state of the art; these sequences are aligned with respect to the Pseudomonas HPPD sequence as the reference sequence and comprise the HPPD sequences as described therein of Streptomyces avermitilis (Genebank SAV 11864), of Daucus carota (Genebank DCU87257), of Arabidopsis thaliana (Genebank AF047834), of Zea mays (Genbank NM001112312), of Hordeum vulgare (Genebank HVAJ693), of Mycosphaerella graminicola (Genebank AF038152), of Coccicoides immitis (Genebank COITRP) and of Mus musculus (Genebank MU54HD). This figure gives the numbering of the amino acids of the Pseudomonas sequence and also the amino acids which are common to these sequences, with these amino acids being designated by an asterisk. On the basis of such an alignment, it is straightforward, from the definition of the Pseudomonas amino acid by its position and its nature, to identify the position of the corresponding amino acid in another HPPD sequence (with the alignment of sequences of different plant, mammalian and bacterial origin demonstrating that this method of alignment, which is well known to a skilled person, can be generalized to any other sequence). An alignment of different HPPD sequences is also described at Maxwell et al. US Patent Publication 20030066102 and Patent Application WO97/49816. The C-terminal part of the HPPDs, which is where the active site of the enzyme is located, differs from its N-terminal part by a linking peptide which ensures the stability of the enzyme and its oligomerization (the Pseudomonas HPPD is a tetramer while the plant HPPDs are dimers). The linking peptide makes it possible to define the N-terminal end of the C-terminal part of the enzyme, with the said peptide being located between amino acids 145 and 157 in the case of Pseudomonas. The C-terminal part can therefore be defined as consisting of the sequence defined, on the one hand, by the linking peptide and, on the other hand, by the C-terminal end of the enzyme, with the mutation which is effected in the C-terminal part of the HPPD therefore being effected in the region which has thus been defined. Two amino acids, which are in positions 161 and 162 in the case of the Pseudomonas sequence (D=Asp161 and H=His162), will be noted in all sequences. With reference to the Pseudomonas HPPD, it is therefore possible to define the linking peptide which represents the N-terminal end of the C-terminal part of the HPPD as being located between approximately 5 and 15 amino acids upstream of the amino acid Asp161. The mutation of interest in the present invention is effected on amino acids being located with reference to the Pseudomonas sequence at position 336.
[0007] There is a need to identify antibodies that are immunoreactive with the mutant HPPD protein described above so that plants containing such mutated HPPD protein can be readily identified. Especially useful would be an antibody that immunoreacts with Pseudomonas HPPD and especially the mutant HPPD protein containing the mutation at residue 336 (glycine (gly or G) to tryptophan (trp or W)). A method that would avoid time-consuming lab steps would reduce costs, allowing for quick identification of the transgenic plants containing the mutant protein, aiding in breeding and selection. Furthermore, antibodies that are immunoreactive with such proteins could be useful in isolating and purifying the proteins.
[0008] All references cited are incorporated herein by reference. The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 22, 2009, is named 210004.txt, and is 12,549 bytes in size.
[0009] The invention is directed to hybridomas and the antibodies and fragments produced from the hybridomas, which are immunoreactive with the amino acid sequence of a mutant HPPD gene. The amino acid sequence is that which substitutes a glycine for tryptophan at residue 336 when compared to a wild-type HPPD polypeptide, and in one embodiment makes such substitution at position 336 of the Pseudomonas fluorescens A32 HPPD gene as is represented at SEQ ID NO: 4 and also with the corresponding mutations shown in FIG. 1 and is SEQ ID NO: 3. Use of the antibodies to identify plant cells having said amino acids and to isolate and purify same are also disclosed.
[0010] FIG. 1 shows the His-tag used in isolating the sequence, which is the first 33 bases in bold (SEQ ID NO: 1) and the 1077 nucleotide sequence of the open reading frame of the HPPD mutant beginning at base pair 34 with the ATG start site (SEQ ID NO: 2) and the encoded amino acid sequence (SEQ ID NO: 3) set forth below. The mutation of glycine to tryptophan is in bold and underlined and corresponds with position 336 of the Pseudomonas fluorescens A32 HPPD (SEQ ID NO: 4).
[0011] FIG. 2 is an SDS-PAGE gel of the mutant HPPD-expressed in E. coli. Lane 1 is supernatant of sonicate (soluble part), lane 2 is precipitate of sonicate (inclusion body) and lane 3 shows molecular weight standards.
[0012] FIG. 3 is an SDS-PAGE gel of mutant HPPD target protein purification profile by Ni-IDS affinity chromatography. Lane 1 is supernatant of sonicate, lane 2 is wash, lane 3 is eluate and lane 4 molecular weight standards.
[0013] FIG. 4A is a Western blot using monoclonal antibody 2E6; 4B is using monoclonal antibody 1C5; 4C is using monoclonal antibody 6H11; and 4D is using the polyclonal antiserum. Lane 1 is the molecular weight standards, lane 2 is cotyledon extract of a transgenic plant expressing HPPD protein, lane 3 is a non-transformed soybean plant with added purified HPPD protein (lane 4 is empty) and lane 5 is cotyledon extract of a non-transformed soybean plant.
[0014] Described here are hybridomas and antibodies and fragments of same, prepared from these hybridomas against a mutant hydroxyphenyl pyruvate dioxygenase (HPPD) enzyme. The monoclonal antibodies immunoreactive with and are useful to identify presence of the enzyme, and to isolate and purify the enzyme. Terms used herein employ their common definitions; for example, immunoreactive refers to reacting to particular antigens or haptens, and wild-type refers to the polypeptide as it occurs in nature. A mutant HPPD enzyme is one in which, compared to a wild-type HPPD, which in an embodiment can be the Pseudomonas HPPD, there is a mutation of the amino acid of the enzyme aligned with position 336 of that HPPD protein as can be readily determined, as described supra. See also, for example, GenBank Accession No. DQ459070, which aligns with the mutant except at this particular position. As the mutant is shown in FIG. 1, the mutation occurs at position 336 (excluding the His-tag) As shown in the examples below, the monoclonal antibody is immunoreactive to a HPPD protein containing tryptophan at residue 336 of the HPPD of P. fluorescens. This sequence is shown in FIG. 1 with the mutation position bolded and underlined and is SEQ ID NO: 3. Reference to the numbering of residues of the HPPD amino acid is used in examples here not to limit the invention, but to facilitate comparison of HPPD sequences from sources from various sources. As used herein when referring to the "antibody" or "polyclonal antibody" or "monoclonal antibody" (MAb) of the invention is meant an antibody or fragment of same that is immunoreactive with a Pseudomonas HPPD amino acid sequence having said mutation.
[0015] An antibody (or an immunoglobulin) is a protein synthesized by an animal in response to the presence of a foreign substance that is called an antigen. Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, but all antibodies have the same overall structure. An antibody molecule is composed of two distinct regions. One is a constant region and the other is a variable region that gives an antibody the specificity to a vast variety of different antigens.
[0016] Five major classes of antibodies are IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant class. IgG, as an example, has a molecular weight of 150 kDa and is composed of two different types of polypeptide chains: one is the heavy chain (50 kDa) and the other is the light chain (25 kDa). Each IgG molecule has two heavy chains and two light chains linked by disulfide bonds. Variable regions of the heavy (VH) and light (VL) chains together function as the variable region of the antibody and give the antibody the ability to bind a specific antigen.
[0017] In the amino acid sequences discussed here, the standard single letter or three letter nomenclature is used. All peptide structures represented in the following description are shown in conventional format in which the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus at the right Likewise, amino acid nomenclature for the naturally occurring amino acids found in protein is as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr,T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V). An "X" may be used when the amino acid residue is unknown and parentheses designate that an unambiguous assignment is not possible and the amino acid designation within the parentheses is the most probable estimate based on known information.
[0018] Deoxyribonucleic acid (DNA) is a polymer comprising four mononucleotide units, DAMP (2'-Deoxyadenosine-5-monophosphate), dGMP (2'-Deoxyguanosine-5-monophosphate), dCMP (2'-Deoxycytosine-5-monophosphate) and dTMP (2'-Deoxycytosine-5-monophosphate) linked in various sequences by 3',5'-phosphodiester bridges. The structural DNA consists of multiple nucleotide triplets called "codons" which code for the amino acids. The codons correspond to the various amino acids as follows: Arg (CGA, CGC, CGG, CGT, AGA, AGG); Leu (CTA, CTC, CTG, CTT, TTA, TTG); Ser (TCA, TCC, TCG, TCT, AGC, AGT); Thr (ACA, ACC, ACG, ACT); Pro (CCA, CCC, CCG, CCT); Ala (GCA, GCC, GCG, GCT); Gly (GGA, GGC, GGG, GGT); Ile (ATA, ATC, ATT); Val (GTA, GTC, GTG, GTT); Lys (AAA, AAG); Asn (AAC, AAT); Gln (GAA, CAG); His (CAC, CAT); Glu (GAA, GAG); Asp (GAC, GAT); Tyr (TAC, TAT); Cys (TGC, TGT); Phe (TTC, TTT); Met (ATG); and Trp (UGG). Moreover, due to the redundancy of the genetic code (i.e., more than one codon for all but two amino acids), there are many possible DNA sequences which may code for a particular amino acid sequence.
[0019] Methods of producing polyclonal antibodies are known to those of skill in the art. Typically, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein of interest. When appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Chapter 5, p. 76, Cold Spring Harbor Laboratory, New York (1988); or Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.
[0020] Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519, incorporated herein by reference). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler and Milstein (1975) Nature 256:495-497, which discusses one method of generating monoclonal antibodies. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. Clearly many such different methods and variations are available to one skilled in the art.
[0021] The use of somatic hybrid cell lines as sources of antibody to individual antigens generally dates from the work of Kohler and Milstein (1975), supra. The antibodies produced are quite different than those recovered from antiserum from conventionally immunized animals. Each hybrid cell line synthesizes a homogenous immunoglobulin that represents but one of the myriad of types of antibodies that an animal can synthesize in response to an antigen in vivo. Since each immunoglobulin-producing clone is characterized by the single type of antibody it produces, the term monoclonal antibody has been adopted. The advantages of monoclonal antibodies are numerous; they can be obtained in large supply; the preparation is homogenous with respect to antigen reactivity and remains so over time.
[0022] The principle of hybridoma/monoclonal technology is predicated on the observation that when two somatic cells are fused the resultant hybrid displays characteristics of both of the parent cell types. In the case of monoclonal antibody production, the ability to synthesize the particular antibody is derived from an immunocompetent cell (usually a spleen cell) taken from an immunized donor animal, whereas the ability to continuously divide in cell culture is contributed by the other fusion partner, a tumor cell line (often a myeloma). Early fusions were complicated by the fact that myeloma cell line also produced a monoclonal antibody; thus the hybrid often produced two types of monoclonal antibody, one of myeloma origin and the other directed by the genetic information of the immunocompetent cell. Subsequently, tumor cells lines incapable of producing their own monoclonal have been used, e.g., SP2/0-Ag14 or X63-Ag8.653, thereby simplifying the analysis of the resultant fusion products.
[0023] Another technical consideration involves the rationale for selecting the successful fusion events (hybrid cells) from the two types of parental cells. Routinely a million or more cells of each type are used in the fusion protocol, and since fusion does not occur with 100% frequency, the job of trying to recover fusion products from the high background of unfused or self-fused parents can be formidable. As mentioned hybridomas are formed by the fusion of short-lived antibody producing (spleen) cells and long-lived myeloma cells. The desired result is a long-lived cell line which produces antibody. Since the spleen cells have a finite life span in culture one can simply wait an appropriate period for all the nonfused or self-fused spleen cells to die; however one must still recover from the resultant population the long-lived antibody producing cells from the long-lived antibody non-producing cells. A popular means for selection hybrid cells is the so-called HAT-selection system. This system involves the use of the enzyme hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). This enzyme functions in the purine salvage pathway in mammalian cells. These cells are also capable of synthesizing purines de novo. Under most conditions, both pathways probably operate to a certain extent. If a cell lacks HGPRT, the salvage pathway is blocked and purines must be manufactured from non-purine materials.
[0024] The chemical 8-azaguanine is an antimetabolite which is capable of masquerading as the purine guanine and replacing it in some of its normal reactions. Azaguanine is incorporated into DNA, interfering with the normal growth pattern and leading to cell death. Since azaguanine must be salvaged, any cell which lacks HGPRT activity cannot utilize azaguanine and will grow in its presence.
[0025] A selective system which operates on the same enzyme but in the opposite sense in that HGPRT positive cells are selected is described by J. W. Littlefield (Science, 145: 709 (1964)). It is called HAT and contains hypoxanthine, aminopterin and thymidine (HAT medium). Aminopterin is an antimetabolite that prevents de novo purine synthesis and methylation of deoxyuridylate to form thymidylate. Hypoxanthine can serve as a salvagable purine in the event that aminopterin blocks de novo purine biosynthesis while thymidine bypasses the necessity for the methylation of thymidylate. Thus, in the presence of aminopterin, any cell with positive HGPRT activity will proliferate while cells with negative HGPRT activity will die.
[0026] In a hybrid system which can be used for selection in accordance with the invention, the myeloma cells are resistant to azaguanine and susceptible to aminopterin, that is, they are HGPRT negative. Thus, they will die in the presence of aminopterin. The antibody producing cells are HGPRT positive. By fusing the cells and growing them in HAT medium without azaguanine (HT medium), the successfully fused cells are selected because the myeloma cells which constitute the proliferating line can only grow where HGPRT activity is present and this activity must be supplied by the HGPRT positive cell line. The antibody producing HGPRT positive cell line are not killed in this medium. They will live for a time but will not proliferate.
[0027] Thus, by fusing the cells in a HAT medium, systems are produced in which the myeloma cells and antibody producing cells can grow long enough to produce hybrid cells but in which only the hybrid cells can survive and proliferate. After selection each hybridoma clone is then screened for the ability to produce the particular antibody of interest.
[0028] A mutant HPPD protein was purified and used as the antigen in the preparation of the HPPD-specific monoclonal antibody. In one embodiment, a preparation process is characterized in that: a) an extract is made from E. coli expressing the mutant HPPD protein, preserved at low temperature by grinding, centrifugation and filtration, b) the extract obtained is enriched in HPPD protein by precipitation in an appropriate solvent, centrifugation and solubilisation of the precipitate obtained in a buffer solution, c) the active protein thus obtained is purified by chromatography and if desired, d) the hybridomas and monoclonal antibodies are prepared from an antigen solution obtained from one of the preparations obtained in paragraphs a), b), and c) above, e) the hybridomas are screened and the monoclonal antibody or antibodies directed specifically against the mutant HPPD are selected.
[0029] In describing the above is not meant to limit the production of the antibodies of the invention to such precise systems; as further methods of such antibody production are developed and optimized they are well within the scope of the invention.
[0030] The monoclonal antibodies so isolated can be used in a variety of ways. As demonstrated below, the antibodies can distinguish between plant tissue containing the mutated Pseudomonas HPPD protein, and those which do not contain this protein. Thus the antibodies can be used to identify those plant cells, tissue and plants which contain the HPPD protein, thus allowing for selection of such plants without destroying the plant nor requiring extensive field tests.
[0031] Kits useful with the invention may take any of a variety of forms and in general provide for obtaining an amino acid containing sample from plant tissue, a support having affixed to it the antibody of the invention capable of forming a binary complex with the HPPD which may be present in the sample, and a binary complex detecting means. The specifics of the kit beyond employing the antibody or fragment immunoreactive properties of the invention are not critical. The method or means of obtaining the amino acid containing sample is not critical, and can take any form, as long as a plant sample is obtained; the detecting means likewise can take any of a myriad of forms, as is well appreciated by one skilled in the art. Detecting means have been used for some time, and can include, for example, biotin, a fluorescent dye, a radio isotope or an enzyme.
[0032] In one embodiment, for example, the monoclonal antibody may be applied to a support structure, such as a test strip. By way of example without intending to limit the application of the invention, the antibody may be used with an immunostrip. One antibody is conjugated to a gold particle and applied to a fiber pad. A second antibody is striped as a line onto a membrane. A strip is assembled in such a way as that a sample pad is placed into the sample extract and the antigen is wicked up the strip, coming in contact with the conjugated MAb and later with the striped MAb. The striped MAb "captures" the antigen-conjugate complex forming a colored line. If no antigen is present, no line forms. A kit would comprise materials required to perform a test for mutant HPPD.
[0033] In the event precise determinations for presence of the mutant are required, other tests are well known to one skilled in the art. By way of example, without limitation, a Western Blot analysis is among the type of test that may be employed. A Western analysis is a variation of the Southern analysis technique. With a Southern analysis, DNA is cut with restriction endonucleases and fractionated on an agarose gel to separate the DNA by molecular weight and then transferring to nylon membranes. It is then hybridized with the probe fragment which was either 32P labeled (radioactive) or non-radio labeled and washed in an SDS solution. In the Western analysis, instead of isolating DNA, the protein of interest is extracted and separated on an acrylamide gel. The protein is then blotted onto a membrane and treated with a labeling substance. See Towbin et al, (1979) "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications" Proc Natl Acad Sci USA 76(9): 4350-4354; Renart et al (1979). "Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure" Proc Natl Acad Sci USA 76(7): 3116-3120.
[0034] Another way the MAbs can be used is in a double antibody sandwich ELISA. The antigen-antibody interactions are similar to those of the immunostrip but take place in the wells of a polystyrene plate.
[0035] The antibodies may also be used in purifying and isolating the HPPD protein. For example, samples containing proteins may be passed through a chromatography column containing the monoclonal antibodies of the invention such that the proteins bind to and are isolated from other proteins in the sample.
[0036] Clearly the antibodies of the invention may be employed in a variety of uses, a few of which are exemplified here, and which are known to those skilled in the art.
[0037] The following are presented by way of illustration and are not intended to limit the scope of the invention.
Isolation of his-Tagged HPPD Protein
[0038] Isolation of the HPPD protein expressed in bacteria used standard protocols as provided by Qiagen, Inc. (The QIAexpressionist®: A handbook for high-level expression and purification of 6×His-tagged (SEQ ID NO: 5) proteins. Fifth Edition, 2003)
[0039] The protocol used in summarized as follows.
Protein Expression
[0040] 1. The E. coli strain was cultured in 1L LB media for 4 hours and then induced by 0.5 mmol/L IPTG for 5 hours at 28° C.
[0041] 2. The cells were harvested by centrifugation and disrupted by sonication.
[0042] 3. The target protein was largely expressed in soluble form (as shown in FIG. 2; lane 1 showing supernatant of sonicate (soluble part), lane 2 showing precipitate of sonicate (inclusion body) and lane 3 is standard molecular weights.).
Protein Purification
[0043] 4. The cells paste was suspended in LEW Buffer and disrupted with sonication on ice. The sonicate was centrifuged at high speed for 20 min. The supernatant was collected and applied to High Affinity Ni-IDA Resin (Cat. No L00223) to purify the fusion protein (as shown in FIG. 2).
[0044] 5. Dialysis the eluate against 1×PBS.
[0045] Results of SDS-PAGE of mutant HPPD target protein purification profile by Ni-IDA Affinity are shown in FIG. 3. Lane 1 is supernatant of sonicate, lane 2 wash, lane 3 eluate, lane 4 shows standard molecular weights.
Production of Antibodies
[0046] BALB/c mice were primed and boosted three to four times with purified bacterially expressed 6×His-HPPD every two to four weeks. Complete and incomplete Freund's Adjuvant from Sigma were used for the priming and the boostings respectively. After two boosts, serum titers were monitored by ELISA. Once the titers were high enough, splenocytes were harvested from the immunized mice and fused with myeloma cells (P3/NSI/1-Ag4-1) using PEG1500 as a fusion agent. The resulting cell fusion products were diluted in hybridoma medium and seeded into 96-well tissue culture plates. After one day, HAT medium was added to the hybridoma cultures. Since then the medium had been changed every three or four days as necessary with HT medium. After ten to fourteen days of culture with selection, screening was initiated by ELISA.
[0047] Antibodies were screened against with purified 6×His-HPPD and 6×His-KLH (`6×His` disclosed as SEQ ID NO: 5). Ninety-six well Nunc Maxi-sorp Immunoplates® (Nunc # 446612, Roskilde, Denmark) were coated by adding 50 μl per well of solution of HPPD and by adding 50 μl per well of 0.5 μg/ml solution of 6×His-KLH (`6×His` disclosed as SEQ ID NO: 5) in coating buffer (BupH.®. Carbonate-Bicarbonate Buffer, Pierce # 28382, Rockford, Ill.) for one hour at room temperature. The coating buffer was removed and the plate was blocked by adding 250 μl per well of blocking buffer (1% Blocker, TM. BSA, Pierce # 37525, in PBS) for two hours at room temperature. 50 μl of hybridoma supernatant were added into the wells and incubated for one hour at room temperature. Wells were then washed four times with PBS/Tween 20. 50 μl of diluted (1:7,000) HRP-conjugated goat anti-mouse Ig (Southern Biotech #1010-05) was added into the wells and incubated for one hour at room temperature. The wells were washed five times with PBS/Tween 20. Anti-HPPD antibodies were detected by adding 50 μl per well of TMB (tetramethyl benzidine) solution (ImmunoPure®. TMB Substrate Kit, Pierce #34021) for 5 to 10 minutes. Plates were read spectorphotometrically at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, Calif.).
[0048] Following the screenings of fusion products by ELISA, antibodies showing specific binding to HPPD were selected.
ELISA Screening
[0049] Monoclonal antibodies derived from hybridomas produced against HPPD were screened using an indirect double antibody sandwich (DAS) ELISA format. Briefly, plates were coated with a polyclonal antibody specific for HPPD. Buffer spiked with varying concentrations of HPPD, extracts of non-GMO soybean (Jack), and HPPD soybean were then incubated in the plate overnight. The plates were then incubated with HPPD specific monoclonal antibodies and then detected with alkaline phosphatase labeled rabbit anti mouse IgG. Monoclonal antibodies showing specificity for HPPD were chosen for further study. Nine antibodies designated 1C5, 2E6, 3C6, 5E9, 6H11, 7A7, 7F10, 8E7, and 12H1 were selected finally for their assay development. Results of one such screening is shown below.
[0050] The designation "PA056R@ 2 ug/ml" refers to the polyclonal HPPD antibody which was coated on the plate at 2 μg/ml. The category "HPPD μg/ml" refers to a standard curve made up from HPPD that was expressed in E. coli and purified.
[0051] Colorimetric response was recorded as optical density (OD) by an ELISA plate reader at a wavelength of 405 nM. This particular assay was read after 60 minutes of substrate development. After further screening, MAbs 1C5, 2E6, and 6H11 proved to have the greatest utility for detecting HPPD.
TABLE-US-00001 HPPD Hybridoma Screen PA056R@ 2 ug/ml 60 min HPPD μg/ml 1C5 2E6 3C6 5E9 6H11 7A7 7F10 8E7 1 ND ND 1.743 1.623 ND ND 1.412 ND 0.250 0.732 1.149 1.194 0.980 0.969 1.473 0.785 0.726 0.063 0.369 0.679 0.671 0.441 0.527 0.800 0.491 0.291 0.016 0.214 0.391 0.314 0.155 0.268 0.414 0.196 0.148 0.004 0.143 0.251 ND ND 0.173 0.248 ND 0.104 Jack-L 0.125 0.189 0.055 0.059 0.147 0.188 0.065 0.092 FG74-L 0.354 0.435 0.317 0.115 0.413 0.422 0.101 0.210
Western Blot Analysis of Selected Antibodies
[0052] Specificity of the reactivity of the antibodies was confirmed by Western Blot Assay. See FIG. 4A-D. Extracts are obtained from soybean plant not transformed with HPPD (referred to as "Jack"--see Lane 5 of the gels in FIGS. 4A-D); as well as lines transformed with HPPD (Lane 2 of the gels of FIGS. 4A-D). Such lines were transformed by particle gun bombardment. (Described generally at Klein, T. M., Arentzen, R., Lewis, P. A. and Fitzpatrick-McElligott, S. (1992) Transformation of microbes, plants and animals by particle bombardment. Biotechnology (N Y) 10, 286-291) with the mutant HPPD gene and also with a double mutant EPSPS glyphosate resistant gene to create a soybean line (See sequence identifier 3 in U.S. Pat. No. 6,566,587 and U.S. Pat. No. 6,040,497). (Lane 1 is molecular weight standards as indicated and having SeeBlue®Plus2 pre-stained marker from Invitrogen, #LC5929 with myosin, phosphorylase, BSA, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin, lysozyme, aprotinin and insulin, B chain.)
[0053] Purified HPPD protein was also added to a non-transgenic soybean (see Lane 3 of the gels in FIGS. 4A-4D). Extract from the identified plants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, blotted onto nitrocellulose membrane and probed with the monoclonal antibody 2E6 (FIG. 4A) 1C5 (FIG. 4B), 6H11 (FIG. 4c) or polyclonal (FIG. 4D. In general, 8-16% BioRad Ready Gel is used in Bio-Rad Mini Protean 3 apparatus at 15 mA/gel until the samples are completely in gel, and then at 30 mA/gel. The electrophoresed proteins in the gels are transferred to PVDF membrane. Detection of immunoreactive proteins is performed using the Zymed Western Blot Kit (#96-9045).
[0054] A protein with an apparent molecular weight of 40 kD was identified by SDS-PAGE and matches the predicted molecular weight of the mutant HPPD protein sequence. The MAbs 1E6, 1C5 and 6H11 are specific for the HPPD in soybean in a similar fashion when purified HPPD is added to non-transgenic plant extract. Bands below the major band are degradation products from the full length protein. This is deduced because they are only detected in extracts containing HPPD and not in the non-transgenic extract. The degradation products are detected somewhat differently by the different monoclonals, as is to be expected since each recognizes a different epitope on the HPPD mutant protein molecule. As is demonstrated in the results, specifically lane 5 of the Westerns in FIG. 4A-D, endogenous non-mutant plant HPPD is not detected by the monoclonals, thus providing the ability to detect presence of the mutant Pseudomonas HPPD using the monoclonal antibodies.
Sequence CWU
5133DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1atgaaacatc accatcacca tcaccatatg cag 3321077DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 2atg gca gat cta tac gaa aac cca atg ggc ctg atg ggc ttt gaa ttc 48Met Ala Asp Leu Tyr Glu Asn Pro Met Gly Leu Met Gly Phe Glu Phe1 5 10 15atc gaa ttc gca tcg ccg acg ccg ggt acc ctg gag ccg atc ttc gag 96Ile Glu Phe Ala Ser Pro Thr Pro Gly Thr Leu Glu Pro Ile Phe Glu 20 25 30atc atg ggc ttc acc aaa gtc gcg acc cac cgt tcc aag aac gtg cac 144Ile Met Gly Phe Thr Lys Val Ala Thr His Arg Ser Lys Asn Val His 35 40 45ctg tac cgc cag ggc gag atc aac ctg atc ctc aac aac gag ccc aac 192Leu Tyr Arg Gln Gly Glu Ile Asn Leu Ile Leu Asn Asn Glu Pro Asn 50 55 60agc atc gcc tcc tac ttt gcg gcc gaa cac ggc ccg tcg gtg tgc ggc 240Ser Ile Ala Ser Tyr Phe Ala Ala Glu His Gly Pro Ser Val Cys Gly65 70 75 80atg gcg ttc cgc gtg aag gac tcg caa aag gcc tac aac cgc gcc ctg 288Met Ala Phe Arg Val Lys Asp Ser Gln Lys Ala Tyr Asn Arg Ala Leu 85 90 95gaa ctc ggc gcc cag ccg atc cat att gac acc ggg ccg atg gaa ttg 336Glu Leu Gly Ala Gln Pro Ile His Ile Asp Thr Gly Pro Met Glu Leu 100 105 110aac ctg ccg gcg atc aag ggc atc ggc ggc gcg ccg ttg tac ctg atc 384Asn Leu Pro Ala Ile Lys Gly Ile Gly Gly Ala Pro Leu Tyr Leu Ile 115 120 125gac cgt ttc ggc gaa ggc agc tcg atc tac gac atc gac ttc gtg tac 432Asp Arg Phe Gly Glu Gly Ser Ser Ile Tyr Asp Ile Asp Phe Val Tyr 130 135 140ctc gaa ggt gtg gag cgc aat ccg gtc ggt gca ggt ctc aaa gtc atc 480Leu Glu Gly Val Glu Arg Asn Pro Val Gly Ala Gly Leu Lys Val Ile145 150 155 160gac cac ctg acc cac aac gtc tat cgc ggc cgc atg gtc tac tgg gcc 528Asp His Leu Thr His Asn Val Tyr Arg Gly Arg Met Val Tyr Trp Ala 165 170 175aac ttc tac gag aaa ttg ttc aac ttc cgt gaa gcg cgt tac ttc gat 576Asn Phe Tyr Glu Lys Leu Phe Asn Phe Arg Glu Ala Arg Tyr Phe Asp 180 185 190atc aag ggc gag tac acc ggc ctg act tcc aag gcc atg agt gcg ccg 624Ile Lys Gly Glu Tyr Thr Gly Leu Thr Ser Lys Ala Met Ser Ala Pro 195 200 205gac ggc atg atc cgc atc ccg ctg aac gaa gag tcg tcc aag ggc gcg 672Asp Gly Met Ile Arg Ile Pro Leu Asn Glu Glu Ser Ser Lys Gly Ala 210 215 220ggg cag atc gaa gag ttc ctg atg cag ttc aac ggc gaa ggc atc cag 720Gly Gln Ile Glu Glu Phe Leu Met Gln Phe Asn Gly Glu Gly Ile Gln225 230 235 240cac gtg gcg ttc ctc acc gac gac ctg gtc aag acc tgg gac gcg ttg 768His Val Ala Phe Leu Thr Asp Asp Leu Val Lys Thr Trp Asp Ala Leu 245 250 255aag aaa atc ggc atg cgc ttc atg acc gcg ccg cca gac act tat tac 816Lys Lys Ile Gly Met Arg Phe Met Thr Ala Pro Pro Asp Thr Tyr Tyr 260 265 270gaa atg ctc gaa ggc cgc ctg cct gac cac ggc gag ccg gtg gat caa 864Glu Met Leu Glu Gly Arg Leu Pro Asp His Gly Glu Pro Val Asp Gln 275 280 285ctg cag gca cgc ggt atc ctg ctg gac gga tct tcc gtg gaa ggc gac 912Leu Gln Ala Arg Gly Ile Leu Leu Asp Gly Ser Ser Val Glu Gly Asp 290 295 300aaa cgc ctg ctg ctg cag atc ttc tcg gaa acc ctg atg ggc ccg gtg 960Lys Arg Leu Leu Leu Gln Ile Phe Ser Glu Thr Leu Met Gly Pro Val305 310 315 320ttc ttc gaa ttc atc cag cgc aag ggc gac gat ggg ttt ggc gag tgg 1008Phe Phe Glu Phe Ile Gln Arg Lys Gly Asp Asp Gly Phe Gly Glu Trp 325 330 335aac ttc aag gcg ctg ttc gag tcc atc gaa cgt gac cag gtg cgt cgt 1056Asn Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Asp Gln Val Arg Arg 340 345 350ggt gta ttg acc gcc gat taa 1077Gly Val Leu Thr Ala Asp 3553358PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 3Met Ala Asp Leu Tyr Glu Asn Pro Met Gly Leu Met Gly Phe Glu Phe1 5 10 15Ile Glu Phe Ala Ser Pro Thr Pro Gly Thr Leu Glu Pro Ile Phe Glu 20 25 30Ile Met Gly Phe Thr Lys Val Ala Thr His Arg Ser Lys Asn Val His 35 40 45Leu Tyr Arg Gln Gly Glu Ile Asn Leu Ile Leu Asn Asn Glu Pro Asn 50 55 60Ser Ile Ala Ser Tyr Phe Ala Ala Glu His Gly Pro Ser Val Cys Gly65 70 75 80Met Ala Phe Arg Val Lys Asp Ser Gln Lys Ala Tyr Asn Arg Ala Leu 85 90 95Glu Leu Gly Ala Gln Pro Ile His Ile Asp Thr Gly Pro Met Glu Leu 100 105 110Asn Leu Pro Ala Ile Lys Gly Ile Gly Gly Ala Pro Leu Tyr Leu Ile 115 120 125Asp Arg Phe Gly Glu Gly Ser Ser Ile Tyr Asp Ile Asp Phe Val Tyr 130 135 140Leu Glu Gly Val Glu Arg Asn Pro Val Gly Ala Gly Leu Lys Val Ile145 150 155 160Asp His Leu Thr His Asn Val Tyr Arg Gly Arg Met Val Tyr Trp Ala 165 170 175Asn Phe Tyr Glu Lys Leu Phe Asn Phe Arg Glu Ala Arg Tyr Phe Asp 180 185 190Ile Lys Gly Glu Tyr Thr Gly Leu Thr Ser Lys Ala Met Ser Ala Pro 195 200 205Asp Gly Met Ile Arg Ile Pro Leu Asn Glu Glu Ser Ser Lys Gly Ala 210 215 220Gly Gln Ile Glu Glu Phe Leu Met Gln Phe Asn Gly Glu Gly Ile Gln225 230 235 240His Val Ala Phe Leu Thr Asp Asp Leu Val Lys Thr Trp Asp Ala Leu 245 250 255Lys Lys Ile Gly Met Arg Phe Met Thr Ala Pro Pro Asp Thr Tyr Tyr 260 265 270Glu Met Leu Glu Gly Arg Leu Pro Asp His Gly Glu Pro Val Asp Gln 275 280 285Leu Gln Ala Arg Gly Ile Leu Leu Asp Gly Ser Ser Val Glu Gly Asp 290 295 300Lys Arg Leu Leu Leu Gln Ile Phe Ser Glu Thr Leu Met Gly Pro Val305 310 315 320Phe Phe Glu Phe Ile Gln Arg Lys Gly Asp Asp Gly Phe Gly Glu Trp 325 330 335Asn Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Asp Gln Val Arg Arg 340 345 350Gly Val Leu Thr Ala Asp 3554358PRTPseudomonas fluorescens 4Met Ala Asp Leu Tyr Glu Asn Pro Met Gly Leu Met Gly Phe Glu Phe1 5 10 15Ile Glu Phe Ala Ser Pro Thr Pro Gly Thr Leu Glu Pro Ile Phe Glu 20 25 30Ile Met Gly Phe Thr Lys Val Ala Thr His Arg Ser Lys Asn Val His 35 40 45Leu Tyr Arg Gln Gly Glu Ile Asn Leu Ile Leu Asn Asn Glu Pro Asn 50 55 60Ser Ile Ala Ser Tyr Phe Ala Ala Glu His Gly Pro Ser Val Cys Gly65 70 75 80Met Ala Phe Arg Val Lys Asp Ser Gln Lys Ala Tyr Asn Arg Ala Leu 85 90 95Glu Leu Gly Ala Gln Pro Ile His Ile Asp Thr Gly Pro Met Glu Leu 100 105 110Asn Leu Pro Ala Ile Lys Gly Ile Gly Gly Ala Pro Leu Tyr Leu Ile 115 120 125Asp Arg Phe Gly Glu Gly Ser Ser Ile Tyr Asp Ile Asp Phe Val Tyr 130 135 140Leu Glu Gly Val Glu Arg Asn Pro Val Gly Ala Gly Leu Lys Val Ile145 150 155 160Asp His Leu Thr His Asn Val Tyr Arg Gly Arg Met Val Tyr Trp Ala 165 170 175Asn Phe Tyr Glu Lys Leu Phe Asn Phe Arg Glu Ala Arg Tyr Phe Asp 180 185 190Ile Lys Gly Glu Tyr Thr Gly Leu Thr Ser Lys Ala Met Ser Ala Pro 195 200 205Asp Gly Met Ile Arg Ile Pro Leu Asn Glu Glu Ser Ser Lys Gly Ala 210 215 220Gly Gln Ile Glu Glu Phe Leu Met Gln Phe Asn Gly Glu Gly Ile Gln225 230 235 240His Val Ala Phe Leu Thr Asp Asp Leu Val Lys Thr Trp Asp Ala Leu 245 250 255Lys Lys Ile Gly Met Arg Phe Met Thr Ala Pro Pro Asp Thr Tyr Tyr 260 265 270Glu Met Leu Glu Gly Arg Leu Pro Asp His Gly Glu Pro Val Asp Gln 275 280 285Leu Gln Ala Arg Gly Ile Leu Leu Asp Gly Ser Ser Val Glu Gly Asp 290 295 300Lys Arg Leu Leu Leu Gln Ile Phe Ser Glu Thr Leu Met Gly Pro Val305 310 315 320Phe Phe Glu Phe Ile Gln Arg Lys Gly Asp Asp Gly Phe Gly Glu Gly 325 330 335Asn Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Asp Gln Val Arg Arg 340 345 350Gly Val Leu Thr Ala Asp 35556PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 5His His His His His His1 5
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Michal Szymanski <mich20061(at)gmail(dot)com> writes: >> Exactly what modifications did you make? > We have added few new columns and we delete one column. [ experiments... ] Hmm, looks like RETURN QUERY isn't too bright about dropped columns in the function result type. I'm not too sure about how hard this is to fix, but in the meantime you will need to dump and recreate/reload that particular table in order to get it to work. regards, tom lane
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Dior Miss Dior Cherie - Shower Gel Reviews
Raspberries & TobaccoMiss Dior Cherie is one of my new favorite perfumes this fall. It smells like raspberries and fresh tobacco to me! MMMMMM. This is very expensive but it feels and smells absolutely amazing. This is a perfect match to the perfume. — 6 years, 10 months agopynkNymphette | 197 reviews
Scent is wonderfulOne of the best scented showers I could ever take. This leaves your skin extra soft and smooth — 7 years, 9 months agoPamela937 | 542 reviews
DiorQuite expensive but so worth it. The scent is heavenly & my skin feels so silky. — 6 years, 10 months agoKARENCOGBURN | 285 reviews
Too strongDidnt wash well and too strong of a fragrance. — 6 years agomaria_longfellow | 66 reviews
Smells wonderful....I love to smell great and I am always on the lookout for new scents and I fell in love with this one it also makes your skin feel great. I recommend it to anyone that likes to smell great and feel great too. — 9 years, 1 month agoag43098 | 45 reviews
great shower gelThis smells great and also feels wonderful — 9 years, 1 month agosuzettenewcomb | 6 reviews
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Before settling on the name Martha & The Vandellas, the group had two record deals prior to finding a home on Motown's Gordy imprint. The act had a much grittier vocal styling than other sweet, sounding Motown girl groups such as The Supremes.
Original Motown lineup: Rosalind Ashford, Annette Beard and Martha Reeves
Previously Known As: The Del-Phis, The Vels
Founded: 1960
Hometown: Detroit
Biggest Hits: "(Love Is Like A) Heat Wave," "Nowhere To Run," "Jimmy Mack" and "Dancing In The Street"
Style: Soul, doo wop, blues, rock
Label: Checker Records, Checkmate Records, Gordy
Bragging Rights: Pre-Martha & The Vandellas members sang background vocals on Marvin Gaye's first hit, "Stubborn Kind Of Fellow." The group was inducted into the Rock & Roll Hall Of Fame in 1995. Their song "(Love Is Like A) Heat Wave" was nominated for a Grammy for Best R&B Vocal Performance By A Duo Or Group. In 1970, they released the song "I Should Be Proud," which protested the war and marked Motown's first political song. Reeves served on Detroit's city council from 2005 to 2009.
The Supremes are the spotlight group this month in Yahoo! Music's Best Soul Girl Groups Of All Time tribute for Black Music Month. A different act will be profiled everyday. The list includes African American women from a variety of genres — R&B, pop, gospel and rap — who were trailblazers and paved the way for those who followed.


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Tony Manero's always the coolest cat in the house. When he hits the dance floor, he's really on fire. And who better to play him than Mr. Cool himself, John Travolta, in one of his best roles ever. Of course, he was much thinner and younger back then, but he was already as charismatic and natural an actor as he is today. And man, what a great dancer he is! You gotta see him getting his groove to The Bee Gees's "You Should Be Dancing"!
Yet the film's about more than disco. It's actually a rather thankless description of teenage life, circa NYC in 77. Manero and his buddies often get into trouble with other gangs like the Barracudas. There's also a guy who knocks up his girlfriend and doesn't know what to do, and even Tony starts questioning his lifestyle. But the dancing is still central to the story. Tony takes part in the 2001's annual dance contest. He first teams up with some bitch whom he soon dumps for Stephanie, a classy Manhattan girl he meets. She's quite a bitch herself, and she doesn't seem to know what she wants, but there's no doubt that she and Manero form a great dancing couple.
The film was directed by John Badham, who's got an interesting style even though he's not, like, Scorsese. His film is truly dynamite. It's as cool as it gets, but it's also smart and well crafted, and the soundtrack is one of the best ever. Definitively a must-see.


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Where6040 N. Pulaski RoadChicago, IL 60646
Habitat for Humanity is searching for outgoing and motivated volunteers to help increase our donation drop-offs at our ReStore. Volunteers will be distributing door hanger flyers throughout preselected neighborhoods. The ideal volunteer for this position enjoys walking and does not mind working independently or in small groups. This opportunity is a perfect match for people who are interested in seeing new/different neighborhoods, want to gain map reading skills or are looking for a low stress way to get involved with us.
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Recyclable materials brought to a Greenhouse Recycling center must be 100% California Redemption Value (CRV), empty, clean and dry. Coupon pricing does not apply to material not meeting these conditions. One unexpired coupon may be used per-visit. We also accept unexpired competitor coupons!


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Sharing my favorite meal preparations, focusing on meals or snacks that are easy to prepare while.. healthy to eat. Neda Firouz
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The life and times of Charlotte in a small Florida town. Her adventures and her daily thoughts.. gathered for you and posted at the lake point.
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Better broccoli, enhanced anti-cancer benefits with longer shelf life
NewsRX Science
By a
And, according to the recently published study, the method is a natural and inexpensive way to produce broccoli that has even more health benefits and won't spoil so quickly on your refrigerator shelf.
"We had figured out ways to increase the anti-cancer activity in broccoli, but the way we figured it out created a situation that would cause the product to deteriorate more rapidly after application," Juvik said. "For fresh-market broccoli that you harvest, it's not too big a deal, but many of these products have to be shipped, frozen, cut up, and put into other products. Usually the idea is to get it from the farm to at least the distributor (grocery store) within two to three days.
"If we could figure out a way to prolong the appearance, taste, and flavor long after harvest and maintain the improved health-promoting properties, that's always of great interest to growers," he added.
The researchers first used methyl jasmonate (MeJA), a non-toxic plant-signal compound (produced naturally in plants) to increase the broccoli's anti-cancer potential, which they sprayed on the broccoli about four days before harvest. When applied, MeJA initiates a process of gene activity affiliated with the biosynthesis of glucosinolates (GS), which are compounds found in the tissue of broccoli and other brassica vegetables (such as cauliflower, cabbage, and kale).
Glucosinolates have been identified as potent cancer-preventative agents because of their ability to induce detoxification enzymes, such as quinone reductase (QR), that detoxify and eliminate carcinogens from the human body.
However, during this process, MeJA also signals a network of genes that lead to plant decay by inducing the release of ethylene, Juvik explained. "While we can use MeJA to turn on phytochemicals like the glucosinolates and dramatically increase the abundance of those helpful anti-cancer compounds, MeJA also reduces the shelf life after harvest," he said.
So the researchers tried using the recently developed compound 1-methylcyclopropene (1-MCP), which has been shown to interfere with receptor proteins in the plant that are receptor-sensitive to ethylene. They applied the compound after harvesting the same broccoli that had already been treated with MeJA before harvest.
"Ethylene will move and bind to ethylene receptors and that binding process initiates decay. What this compound does is that it more competitively lands on the protein and binds to or pushes out ethylene," Juvik explained. "It basically stops or dramatically slows down the decay associated with ethylene.
"The combination is good," he said.
Like MeJA, 1-MCP is also a non-toxic compound naturally produced in plants, although Juvik said synthetic forms can be produced. He stressed that both the MeJA and 1-MCP treatments required very small amounts of the compounds.
"It's very cheap, and it's about as toxic as salt. It takes very little to elevate all the desirable aspects. It's volatile and disappears from the product after about 10 hours," he said.
The use of these treatments could make a great impact on important global dilemmas such as food security issues and health-care costs, Juvik said.
"It's a fairly cheap way to maintain quality, but it provides a preventative approach to all the medical costs associated with degenerative diseases. These are not pills that go in and take away or change damaged tissues, but it's a way to protect people by reducing the risk they currently have to different diseases. It won't take it away, but it could prevent further damage," he said.
As for its impact on impending global food security concerns, Juvik said any mechanisms that will improve people's health, especially later in life, will benefit food security.
"We need to look at what mechanisms we can use to improve not only food security but the functioning of people later in their life spans. When you look at how much
Keywords for this news article include: Alkenes, Ethylenes, Glucosinolates, Thioglucosides,
Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


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After the recent hubbub of the Grammys, the sad demise of Whitney Houston, and the surrealistic news of further recorded collaborations by contemporary singing stars Rihanna and Chris Brown, one might say...anything goes!
And so, to celebrate, to get in the mood--to, in a sense, cleanse my palate of all that today's music scene has to offer--this week I decided to get rid of all my preconceived notions of what was right, wrong, good or bad, and, indeed, start from scratch! I would listen to every new piece of music that crossed my path and judge it purely for what it was--without prejudice, critical snobbery, or that numbing sense that everything now being done was once done before, but, sadly, even better.
And thus, to begin my quest, I decided I would start anew by listening to the complete recorded works of Dutch rockers Golden Earring, a long-lived band who since the mid-'60s have recorded a fascinating catalog of slightly off popular music that would eventually encompass a fascinating side-long cover version of the Byrds' "Eight Miles High," a memorable testosterone-infused hit with "Radar Love," a staggering classic via the accompanying "Candy's Going Bad," an early MTV hit with "Twilight Zone," and a whole bunch of other albums that, remarkably, have never stopped coming. All over the place.
In short: They are now the critical baseline by which I will now render all of my judgments regarding today's popular music!
Were I not to notify you of this, I would be doing you a disservice!
Sleigh Bells: Reign Of Terror (Mom + Pop) While I am sadly hip enough to know this trendy duo appeared on Saturday Night Live this weekend, I simply did not have the energy to watch their performance! And it didn't matter! Because between Twitter and Facebook, enough people would tell me exactly what I needed to think--and I could spend my Saturday night listening to the complete works of Dutch rockers Alquin and, as is my goal, eventually hear everything! But I like this record, I like the aesthetic notion of loud and distorted guitars, a pleasing but mildly off-pitch female vocalist singing the barely understandable lyrics of "Road To Hell," hot babes, and album covers featuring bloody tennis shoes! For that matter, both the band name and the album title bring to mind the notion of an evil Santa Claus, and that's not a bad thing! By following no formula whatsoever, Sleigh Bells are an interesting, unique, noisy outfit that are infinitely more compelling than a night's worth of erectile dysfunction commercials--and almost up there with the charismatic cast of characters populating today's fascinating Mucinex spots! Bold words? Perhaps!
The Chieftains: Voices Of Ages (Hear Music/Concord) Aged, ancient and pretty spry, the Chieftains continue to roll out highly credible excursions of traditional Irish music, and this latest effort--which marks their 50th anniversary--should bring in a welcome slab of today's youth market, what with inclusion of such collaborators as Bon Iver, the Decemberists, the Civil Wars, the Punch Brothers, the Pistol Annies, and ultrahipster NASA Astronaut Cady Coleman, last seen cavorting at the Electric Daisy Festival, careening around shirtless at Burning Man, and looking for a similar verb starting with "c" to associate with another completely unlikely event purely for the sake of pathetic humor! A tasteful, well-played venture, certain to eventually cement the distinguished band's reputation as "those old guys who played with other, hipper guys again and again to broaden their commercial reach" according to today's poor kids forced to hear their dads and moms play this stuff for them in the SUV because that's what good parenting's all about! Bad parents: buy Lucky Charms and play hiphop--it'll all work out!
Sinéad O'Connor: How About I Be Me (And You Be You)? (MRI) This represents the ninth studio album by plucky Irish vocalist O'Connor--these days best remembered as the zany lady who once ripped up a picture of the Pope on national TV, who once had her biggest hit ever with a song she didn't write, and who once had her shaven-headed publicity photo filled in with a black Sharpie by a music writer who said, I forget to whom, "Hey, you know what? With hair, Sinéad O'Connor is actually pretty hot!" This latest album is about as good as any of them were--none of them were exceptional--but, as was the case with I Do Not Want What I Haven't Got, the title seems almost there but sort of misses the mark; were it to be, say, How About I Be Me (And You Be A Nearby Inanimate Object Like, Say, That Chair)?, I, and indeed all society, might find it more compelling! Still, she's hip enough to cover John Grant's "Queen Of Denmark," so let's just call her a goddess and be done with it! Incidentally, speaking as a journalist, why have I only ever used the word "plucky" to describe Sinéad O'Connor?
Isidore: Life Somewhere Else (Communicating Vessels) A very nice, subtle return for odd-man-out collaborators Jeffrey Cain of Remy Zero and Steve Kilbey--the distinguished songsmith who's helmed Australian band the Church all these years, and whose voice and lyrical approach makes this one of his best efforts in recent memory. Atmospheric, thoughtful, intimate and biting, the 14-track set moves along, sonically adventurous and less deliberately droning than has been the latter-day Kilbey norm, and is welcome evidence that our better artists continue to grow with each passing year. Recommended.
Santana: Live At Montreux 2011 (Blu-ray) (Eagle Rock) Aside from the excellent musicianship that has marked every project Carlos Santana has had a hand in, the legendary guitarist's keen understanding of jazz improvisation--the legacy of greats like John Coltrane and Miles Davis--has made every new reworking of past glories an interesting, inspiring and unique listen. This new DVD, shot only last year, offers over 200 minutes of the guitarist and his superb band running through well-known hits such as "Black Magic Woman, " "Evil Ways" and "Oye Como Va," but each time bringing something new to the mix in terms of notes and emotional approach. Featuring guest stars like Susan Tedeschi and Derek Trucks, the disc focuses wholly on inspirational music and, not incidentally, is a fine greatest hits set as well. The number of artists with this lengthy a legacy--and this credible a legacy--still out there performing is dwindling, and for Carlos Santana, this is one triumphant showing.
Cursive: I Am Gemini (Saddle Creek) I'm digging the latest Cursive album, one of Saddle Creek's more interesting artists, largely because of the their sonic versatility--the albums really don't all sound alike--and the fact that they've managed to stay interesting and listenable for seven albums now. This new set recounts the story of twin brothers Cassius and Pollock, twin brothers at odds with the world for supplying them names close but not close enough to be the Castor and Pollux of Greek and Roman mythology, as well as completely understandable vitriol directed at their parents, who apparently had no compunctions about naming one of the boys after a fish! Hey, when my wife was pregnant, I thought about naming our first kid Ba'al, but then I grew up! Buy this for kicks!
Eddie Hazel: Game, Dames & Guitar Thangs (Real Gone Music) If you were a record-buying human who visited record stores on a regular basis during the '70s, it would not be an exaggeration to note you probably saw a new Parliament-Funkadelic "project" every three months or so. George Clinton's impressive and productive legacy provided the world with a memorable array of albums, offshoots, and side-projects--Parlet, the Brides Of Funkenstein, Mutiny, and much, much more--but none have developed the cult following that has this album, by Clinton guitarist Hazel, which came and went, briefly, but affected those who heard it deeply. Rescued and reissued, the album sounds even better than it did in 1977--when it was just part of the assembly line--and can now be appreciated as the cultural touchstone it clearly is. Exemplary musicianship, whacked-out covers of "California Dreaming" and "I Want You (She's So Heavy)," the album is a precious slice of another place and time that now seems far, far away. Fabulous stuff.
Lambchop: Mr. M (Merge) The very unusual, and very compelling, recorded work of Kurt Wagner and his band Lambchop shows no sign of slowing well into its second decade; this excellent disc, the band's 11th, shows the same idiosyncratic subtleties that have made the band one of pop music's more unique and literate aggregations. Blah, blah, blah. Suffer migraine headaches? Plagued by horrendous hangovers? This is the band for you!
Tyga: Careless World: Rise Of The Last King (Young Money) An admittedly out-there concept album by Travie McCoy's rapping cousin Tyga, this new album is based on a memorable game of checkers played by Tyga and his good friend Winnie, the famous Pooh, in which both players watched in stunned amazement as a double-decked pair of checkers rose from the checkerboard, flew out to the kitchen and checked a pager lying by the toaster, and then promptly returned to the board without uttering a word! Heck, I'd make an album if I ever saw that happen! Featuring a batch of superstar guests like Lil Wayne, Nicki Minaj, Drake, Nas, Chris Brown, Robin Thicke, and Jerry Mathers, the album is literally the finest product mankind has ever produced! Plus, it doesn't sound bad!
Fun.: Some Nights (Fueled By Ramen) From the get-go, Fun. has devoted their entire career to refuting those things many Stooges fans hold dear--and here, boldly boasting of even greater virility, they take on a classic Eagles track! Still, putting an engagement ring on your fiancée's toe is pushing it, dudes!


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The Vitamin Serum from skinChemists nourishes, plumps and brightens your skin to keep it looking radiant, healthy and youthful. Formulated with high doses of vitamins A, C, D and E, it provides deep hydration, increases your skin's recovery against everyday damage and supports your skin's natural collagen levels and microcirculation to produce an anti-ageing effect. Your skin's elasticity and texture will be dramatically improved to leave it looking smooth and radiant. - L.M.
Directions for Use: Apply the serum in circular motions onto the desired area in the morning before moisturising, and in the evening after your normal cleansing routine.


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Frimpong out for the season
Emmanuel Frimpong has been ruled out for the rest of the season after rupturing the anterior cruciate ligament in his right knee in Wolverhampton Wanderers' match at QPR.
Frimpong, on loan at Wolves from Arsenal, suffered a similar injury to his left knee in 2010, which kept him out for the season. No date has yet been put down for his return.
The midfielder was withdrawn 23 minutes into the victory at Loftus Road. He made five appearances for Wolves, making one assist.
Wolves said on their official website: "Emmanuel Frimpong is returning back to Arsenal after it was confirmed he ruptured the anterior cruciate ligament in his right knee in Saturday's Premier League game at QPR.
"Frimpong, who had been due to stay on loan at Molineux until the end of the season, will now see specialists in London over the next few days before having surgery and starting a programme of rehabilitation.
"The midfielder, who suffered the same injury to his left knee in 2010, had made an impressive start to his temporary spell at Wolves, catching the eye during his five appearances in gold and black.
"Frimpong is expected to be out for a number of months and will therefore miss the remainder of this season. Everyone at Wolves would like to thank the midfielder for his efforts whilst at the club and join Arsenal in wishing him a swift recovery."
The player said on Twitter: "Bad News is that I'm out for 6-9months with my Knee Gd News is that I will therefore have all the time in my hands to be doin #frimmyfacts. I wanna thank all the wolves fans for their support since I've been here and really Hope wolves stay up this season. I also wanna ask you to pray for my cruciate injury as I will be taking all the punishment it deserves as he or she have been very naughty:)"
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Lord Vamana is the fifth incarnation of Srihari Vishnu. In Vamanavatara (Vaman Avatar), Lord Vishnu appeared as a dwarf Brahmin before King Bali. As a result of this incarnation, demon King Bali got Moksha and the company of Lord Vishnu during Chaturmasa period. Indra, the king of Devas, got back his kingdom.
Story of the Birth of Lord Vamana
This story of the birth of Lord Vamana was sent to us by Narahari Sumadhwa.
When Devataas (Demi gods) lost their power to Balichakravarthi (King Bali), Adithi, the mother of Devataas requested Kashyaparu (Sage Kashyapa) to get a son who can bring back the powers to Devataas from Daithyaas (Demons or Asuras).
Balichakravarthy in his quewt for power of Indra, was doing the 100th Ashwamedha Yagna. He had already completed 99 yagnas. If he completes the 100th yagna he would be eligible for the post of Devendra – he can throw out Indra. (The present Indra is Purandara).
Kashyapa Rushi told Aditi to do Payovratha for one year, which she did successfully. As a result of her Jnaana, bhakthi, vairagya, was born Vamana, on Bhadrapada Shudda Dwadashi in Shravana Nakshatra, and Abhijin Muhoortha. This particular dwadashi is also called as Vijaya Tithi.
When he was born itself, Vamana was having Chaturbhuja, Shanka, Chakra, Gada, Padma. He was neelameghashyama and was having Srivatsa Lanchana.
Aditi-Kashyaparu did the Jatakarma Samskara.
Surya did the Gayatri Mantropadesha.
Bruhaspatyacharyaru gave Yagnopaveeta, kashyaparu gave the "darbe Ududaara", Bhoodevi the Krishnajina, Chandra gave Muttugada Danda (palasha).
Aditi gave him koupeena. Brahma gave Kamandalu, saptarshees gave darbe, saraswathi the aksharamale., Kubera Bhikshapatre, annapoorne gave the first bhikshe.
Vamana after Upanayana, did the Agnikaarya and after this Vamana went to Balichakravarthi's Yagna Shaale in Brukukutsa kshetra, near Narmada River north bank.
Bali Chakravarthy on seeing the Baala Brahmachari Vamana, did the paadapooja of the brahmachari and asked him what he wanted? the land, Gold, Mrustanna? marriage, Elephants, Horses, Chariot, etc?
Vamana just asked him to give three steps of land. Bali was surprised for having asked that little daana. Vamana was satisfied with three steps only. Bali agreed.
Vamana in his first step occupied the entire bhoomandala, and in the second he occupied the sky. No place was left to place the third. Asked Bali where shall he keep the third? Bali replied to put on his head.
That is how Vamana recovered for Devendra all the lost properties from Bali and gave it to Devategalu.
Written by Narahari Sumadhwa


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CYNTHIA BAILEY presents
Supermodel, Real Housewives of Atlanta Star, and CEO of The Bailey Agency School of Fashion Cynthia Bailey is preparing to unveil her first pageant:
The mission of Miss Renaissance Pageant 2012 is to inspire young girls to love, respect and empower themselves in every aspect of their lives. The pageant will serve as a stage to showcase strong, smart, beautiful, and community-minded youth and teens. A portion of the proceeds will go to one of Bailey's favorite non-profit organizations – Hosea Feed the Hungry and Homeless.
The pageant will take place on Saturday, September 8 and Sunday, September 9, at the Georgia International Convention Center in Atlanta. Bailey is looking to select our community's newest teen leaders of change, inspiration and impact. Pageant divisions include: Young Miss Pre-Teen (ages 10 to 12 years old), Junior Miss Teen (ages 13 to 15 years old), and Miss Teen (ages 16 to 18 years old). The deadline to enter is September 3.
The "Cynthia Bailey presents Miss Renaissance Pageant 2012" will be filmed.
For more information regarding competing or attending the event, please visit us at "http://www.MissRenaissancePageant.com" www.MissRenaissancePageant.com or contact The Bailey Agency School of Fashion at 404/622.1791.


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San Francisco, California: Two US men have been found guilty of stealing an American company's secret recipe for making a chemical used to whiten products from cars to the middle of Oreo cookies and selling it to a competitor controlled by the Chinese government.
The four-man, eight-woman federal jury found Robert Maegerle, 78, and Walter Liew, 56, guilty of economic espionage and each could face 15 years or more in prison and hundreds of thousands of dollars in fines.
Noting Liew's connections to China, US District Judge Jeffrey White ordered him taken into custody immediately.
"He has received millions of dollars from the People's Republic of China that remains unaccounted for," Justice White said.
"That's a lot of money that could help someone flee."
The two men were convicted in San Francisco of stealing Delaware-based DuPont Co.'s method for making titanium dioxide, a chemical that fetches $US17 billion ($A18.98 billion) a year in sales worldwide.
Federal prosecutors said Walter Liew and his wife Christina Liew launched a small California company in the 1990s aimed at exploiting China's desperate desire to build a DuPont-like factory to make the chemical.
The couple recruited former DuPont scientists with the single-minded goal of winning Chinese contracts. Maegerle worked for DuPont from 1956 to 1991 before joining the Liews. Prosecutors said he provided the Liews with detailed information about DuPont's Taiwan factory.
Prosecutors say Mr Liew received more than $US20 million from the Chinese company Pangang Group for efforts started in the 1990s to deliver the recipe to China.
Mr Maegerle remained free on bond pending his sentencing scheduled for June 10. He declined comment as he left court. He could face a maximum of 15 years in prison.
The defendants had argued that the Chinese obtained the information from public sources, such as expired patents, and nothing was stolen from DuPont.
However, DuPont spokesman Daniel Turner said "the verdict underscores the consequences faced by those who steal trade secrets".
In addition to economic espionage, Liew was also found guilty of witness tampering, filing false tax returns and bankruptcy fraud. He could face a maximum of 20 years in prison.


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|UNCG Home | IPC Home | IPC eNewsworthy | Contact us
|Volume 11 Edition 7: April 2012. Zachary Dayhuff, Editor
Visitors and Scholars!
During his visit, President Jabir had the opportunity to meet with Dr. William Wiener, Dean of the Graduate School, Jerry McGuire, Associate Vice Chancellor for Economic Development, and Dr. J. Alan Boyette, Vice Provost, as well as several IPC staff, including Dr. Penelope Pynes, Associate Provost for International Programs, Michael Elliott, Director of International Student and Scholar Services, and Dr. C.K. Kwai, Director, UNC Exchange Program.
President Jabir also had the opportunity to meet with Dr. James Ryan, Dean of the Joint School of Nanoscience and Nanoengineering (JSNN), and to tour the JSNN with Dr. Daniel Herr, Nanoscience Department Chair.
Special thanks to everyone who made this a fruitful and engaging visit!
During her visit, Mrs. Verhey-Laan met with a number of faculty and staff, including the following Chairs: Dr. Charles Bolton, History, Dr. Kevin Lowe, Business Administration, Dr. Christopher N. Poulos, Communication Studies, Dr. A Lawrence Jenkens, Art, and Dr. Ann Wallace, English. Mrs. Verhey-Laan also had the opportunity to meet with other faculty, including Dr. Ann Dils, Director of Women's and Gender Studies, Dr. Christian Morary, Professor of American Literature, and Dr. Alexandra Schultheis More, Director of Undergraduate Studies at the Department of English. Mrs. Verhey-Laan also had occasion to meet Dr. Penelope Pynes and Dr. C.K. Kwai, as well as Denise Bellamy and Tom Martinek, Jr., Director and Assistant Director, respectively, of Study Abroad and Exchanges.
Mrs. Verhey-Laan's visit was a great opportunity for international relations at UNCG, and special thanks go out to everyone who made it a successful one.


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Support » Plugin: Shortcode Shortcode » Useful for plugin documentation
3 years, 6 months ago
I included shortcode syntax on a post to document my tabby responsive tabs plugin using html entities for the square brackets so I could show shortcode syntax without the shortcodes being processed.
Switching between the text editor to the visual editor caused the html entities to became converted into square brackets turning the example shortcodes into real shortcodes. These were converted into tabs by the tabby plugin turning my post into a confusing mess.
I created this plugin to prevent this from happening again. The intention was to make a safe way to display example shortcode syntax on the frontend that will never be processed by WordPress.
There is documentation on the codex which outlines the double bracket notation to enables shortcodes to be displayed, but the plugin may still be useful as it adds a span with a class that can be targeted in the stylesheet.


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Hiroaki Miyajima
Hiroaki Miyajima was born in Nagano, Japan, in 1956. He received the M.D. degree (summa cum laude) and the Ph.D. degree from the Hamamatsu University School of Medicine (HUSM), Hamamatsu, Japan, in 1981 and 1989, respectively. Since 1981, Miyajima has been with the Medical and Neurological Laboratory of HUSM. In 1996, he was a Research Associate at HUSM. From 1999 to 2009, Miyajima was an Associate Professor of the Department of Medicine at HUSM. Presently, he is a Professor. He is mainly interested in neurodegenerative disorders associated with iron and copper metabolism, especially aceruloplasminemia. In 2002, he had the Outstanding Abstracts Award of 26th International Congress of Internal Medicine. Also he was awarded a Silver Medal of Video Olympics, Movement Disorders Society's 13th International Congress of Parkinson's Disease and Movement Disorders. Miyajima has been a BioIron Member since 2003.
Biography Updated on 16 January 2011


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For three states, share of Hispanic population returns to the past
'Mexican,' 'Hispanic,' 'Latin American' top list of race write-ins on the 2010 census
Latinos are not the only group of Americans who utilize the "some other race" category on the census form—but they are the most likely to do so. In 2010, 6.2% of Americans selected "some other race," up from 5.5% in 2000. Among all those who answered the race question this way in 2010, 96.8% were Hispanic.
Women's college enrollment gains leave men behind
Even though college enrollment rates among young people have risen in recent decades, a Pew Research Center analysis of U.S. Census Bureau data shows that females outpace males in college enrollment, especially among Hispanics and blacks.
Asian Americans split on whether U.S. immigration system works or needs a major overhaul
45% of Asian Americans say the U.S. immigration system "works pretty well and requires only minor changes" while 47% say the system "needs to be completely rebuilt" or "needs major changes."
In 2014, Latinos will surpass whites as largest racial/ethnic group in California
Hispanics prioritize legalization for unauthorized immigrants over citizenship
46% of all Hispanics and 59% of Hispanic immigrants say they worry "a lot" or "some" that they themselves, a family member or a close friend could be deported. Hispanics alone comprise about three-fourths of all immigrants living in the U.S. illegally.
Republican Chris Christie captures about half the Latino vote
In his landslide re-election victory last night, New Jersey Republican Governor Chris Christie carried 51% of the Latino vote, a 19 point increase from his performance in 2009, according to exit polls.
5 demographic realities behind the creation of Univision/ABC News' "Fusion" channel
Fusion's launch reflects several demographic trends among the nation's 53 million Hispanics, which make up 17% of all Americans.
Hispanic or Latino? Many don't care, except in Texas
Which do you prefer? It's a question Hispanics and non-Hispanics alike have struggled with…except in Texas.
High rate of deportations continue under Obama despite Latino disapproval
The Obama administration has provided a way for young unauthorized immigrants brought to the country illegally as children to remain in the U.S., but the total number of deportations of unauthorized immigrants continue at near record levels.


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I'm running a large database ( data dir is ~9gigs ) on Fedora Core 2 with 7.4.5-PGDG rpm's When using the -Fc dump method + pg_restore, I get: -bash-2.05b$ pg_restore -Fc -d foo -L backup.list /tmp/02\:43-postgresql_database-foo-backup pg_restore: ERROR: invalid input syntax for type timestamp: "52.233334" CONTEXT: COPY foo_data, line 42529, column mod_date: "52.233334" pg_restore: [archiver (db)] error returned by PQendcopy When using pg_dump + psql: I get a similar error. In one table that has about 5.4 million rows, the dump has several incomplete rows in the large block of copy data. It seems to attempt to fill the copy with data from the *next* line. ERROR: invalid input syntax for type timestamp: "4" CONTEXT: COPY foo_data, line 169371, column mod_date: "4" going to 169371 lines after the start of the copy, indeed there is a short row (by two), and two fields in on the *next* line is indeed a "4" Both seem to have the same problem - incomplete COPY row data. any ideas? [ \ / [ >X< Christian Fowler | spider(at)viovio(dot)com [ / \ http://www.viovio.com | http://www.tikipro.org
Next: From: Joshua D. Drake Date: 2004-10-18 15:53:21 Subject: Re: large dumps won't restore Previous: From: Bruce Momjian Date: 2004-10-18 04:08:21 Subject: Re: Longest name for table/column/key/index


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When theres something bland, all upon your plate and the taste is bland so you need a handwho you gonna call?! Ass Blaster! When a little bit of chilli just wont do, when what you need is a serious blast of the good stuff to flush out your system and make that nose run; you need a dose of this horrifically hot Ass Blaster Hot Sauce.
Ideal for adding a kick to any meal, to separate the men from the boys! This sauce comes encased in its own wood-inspired outhouse case so that it can be kept out of the hands of little ones and so that everyone knows just how dangerous this bad boy can be. Made from pure ground habanero chillies, this ass blaster sauce does what it says on the label.
We promise you, youll be spending most of the day after the chilli-morning on the loo!
- Extremely hot Ass Blaster Sauce
- Made from the hottest of Habanero Peppers
- Added Capsicum extract for that added hot kick
- Comes presented in a wooden Outhouse box
- Ideal gift to separate the men from the boys
- 5oz
- 21.0cm x 8.5cm x 8.5cm
Ass Blaster Hot Chilli Sauce Customer Reviews
Related products in Chilli Gifts
Frequently Asked Questions
Do you have an un-answered question about this product? Simply ask us a question and we'll do our best to help.


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HEBRON ESTATES -- Residents have long complained about the drag strip more commonly known as Burkland Boulevard.
Police officers from Pioneer Village have run radar in the past.
But the speeding, as well as the complaints, continued.
As a result, the Hebron Estates City Commission opted in June to have a three-way stop created at the intersection of Burkland Boulevard and Scenic Trail.
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17962 Highland Pines Road, Presque Isle 49777
See more homes for sale in Presque Isle, MI
This property has been Sold!
10/05/2016 at $108,250
Just like NEW! This home has been completely remodeled and is move-in ready. Enjoy 6.3 wooded acres, barn, close to Grand Lake and Lake Huron. Shaded porch and sunny deck. Attached garage. Basement ready to be finished. Private but on a paved road. Make this home your own!
Property Information
- Status
- Sold
- Address
- 17962 Highland Pines Road
- City
- Presque Isle
- Zip
- 49777
- County
- Presque Isle
- Township
- Presque Isle
- Possession
- Negotiable
- Subdivision
- T34n R8e
- Total Finished SqFt
- 2,016
- Garage
- 1.5
- Garage Desc.
- Detached/HTD
- Waterfront Desc
- Public Access
- Water
- Well
- Sewer
- Septic
- Year Built
- 1997
- Home Style
- Ranch
- Taxes
- $2,187
Rooms and Land
- MasterBedroom
- 11x13
- Bedroom2
- 13x9 1st Floor
- Bedroom3
- 11x9 1st Floor
- Dining
- 9x10 1st Floor
- Kitchen
- 11x10 1st Floor
- Living
- 14x17 1st Floor
- Basement
- Full
- Cooling
- Baseboard, Hot Water, Propane
- Heating
- Baseboard, Hot Water, Propane
- Acreage
- 6.3
- Appliances
- Freezer, Range/Oven, Refrigerator
- Interior Features
- Doorwall, Smoke Detector, Vaulted Ceilings
- Exterior Materials
- Vinyl
- Exterior Features
- Barn, Deck, Hunting Prop, Natural, Workshop
Listing Video for 17962 Highland Pines Road, Presque Isle MI 49777
Mortgage Calculator
Listing Broker
Listing Courtesy of Lakeshore Realty
Office Address 7750 E Grand Lake Road
Listings last updated: . Some properties that appear for sale on this web site may subsequently have been sold and may no longer be available.
Our Michigan real estate agents can answer all of your questions about 17962 Highland Pines Road, Presque Isle MI 49777. Real Estate One, Max Broock Realtors, and J&J Realtors are part of the Real Estate One Family of Companies and dominate the Presque Isle, Michigan real estate market. To sell or buy a home in Presque Isle, Michigan, contact our real estate agents as we know the Presque Isle, Michigan real estate market better than anyone with over 100 years of experience in Presque Isle, Michigan real estate for sale.
The data relating to real estate for sale on this web site appears in part from the IDX programs of our Multiple Listing Services. Real Estate listings held by brokerage firms other than Real Estate One includes the name and address of the listing broker where available.
IDX information is provided exclusively for consumers personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
The data relating to real estate one this web site comes in part from the Internet Data Exchange Program of the Water Wonderland MLS (WWLX). Real Estate listings held by brokerage firms other than Real Estate One are marked with the WWLX logo and the detailed information about said listing includes the listing office. Water Wonderland MLS, Inc. © All rights reserved.


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this is a test description
Choose from motel rooms or private cottages when you stay at Georgia Boy's Fish Camp. The motels feature clean, comfortable accommodations with HBO, a twin and queen bed and a coffeemaker. Cottages are available with one or two bedrooms, or as efficiencies, and offer amenities like bed linens and a complete kitchen. And when it comes to fishing, pick up your license here and head out in search of crappies, catfish, bass and more!
Connect the generations the old-fashioned way with a fishing trip to Georgia Boy's Fish Camp! It's the perfect place for your family reunion.
Cast your line for a day of great fishing! The water of Crescent Lake is full of bass, bream, catfish, speckled perch, and so much more.
Group Information
Nearest Airports
- Gainesville Regional Airport (GNV) / 40 miles
- Daytona Beach International Airport (DAB) / 43.5 miles
- Orlando Sanford International Airport (SFB) / 59.8 miles
* Not all airports listed here may be open to public use, or service commercial flights
Traveler Reviews


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Small bug + solution pgAdmin III 1.12.0 PostgreSQL 9.0 Ubuntu Linux 10.04 installed completely from Martin Pitt's PPA https://launchpad.net/~pitti/+archive/postgresql, but also reproducible with original Ubuntu repositories (PostgreSQL 8.4, pgAdmin III 1.10.2) Scenario: 1) start pgAdmin from console: $ pgadmin3 2) warning appears on console: WARNING: password file "/home/mase/.pgpass" has group or world access; permissions should be u=rw (0600) or less 3) now try to connect some server by double-clicking server node in tree, error window appears and server is not connected: An error has occurred: Error connecting to the server: fe_sendauth: no password supplied 4) subsequent tries works, until pgAdmin close Solution: When I change permissions for .pgpass file as warning suggested, connection error for first connect upon pgAdmin start disappears and everything works like a charm. It looks like there is some lack in reading passwords from .pgpass when warning is shown. Keep up good work, guys! Martin Schayna
Next: From: Boblitz John Date: 2011-03-04 06:54:32 Subject: pgadminIII Configuration problem Previous: From: Ashesh Vashi Date: 2011-03-03 04:27:14 Subject: Re: paste into SQL editor from Eclipse on Mac doesn't work


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Director of Research and Enterprise
Professor Liz Towns-Andrews
Tel: 01484 473169
E-mail: firstname.lastname@example.com
Liz joined the University of Huddersfield in September 2009. The key aspect which attracted her to the University and made the biggest impression was the combination of the University's vision and ambition.
A trained chemist, Liz graduated with a PhD in X-ray crystallography before joining the Research Council system. She also has an MBA and is a Fellow of the Institute of Physics.
Her first position was as a scientist at the UK's first synchrotron radiation source at Daresbury in Cheshire working closely with UK academics. At that time, her research included time resolved diffraction of biomolecular and polymeric systems but also required her to design and commission large scale equipment to carry out this work.
Prior to arriving at the University, her role was Director of Knowledge Exchange for the Science and Technology Facilities Council, developing the organisation's strategy for Knowledge Exchange and Economic Impact. During this time she has gained extensive experience of working with industry, of providing hi-tech services, the commercialisation and exploitation of IP and of taking forward public/private partnerships. She was a key person responsible for the establishment of the two national Science and Innovation Campuses at Harwell and Daresbury.
Liz enjoys working at the interface between academia and industry and hopes her in-depth knowledge of the Research Council system and wide-ranging networks with industry, government departments and regional agencies will be of value to the University.
Liz has been instrumental in securing European funding to create the new 3M Buckley Innovation Centre – a flagship hub, supporting spin-in and spin-out companies, linked to the University, providing access to markets, finance, skills and technology. The Centre will act as a catalyst to promote Business to Business and Business to Higher Education collaborations. Liz has been recently appointed the 3M Professor of Innovation at the University and in 2013 was awarded the Queens's Award for Enterprise Promotion, recognising individuals who have played an important role in promoting enterprise skills and supporting entrepreneurs.


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Maybe J.K. Rowling can call her sixth book "Harry Potter and the Four-Fingered Tourists." That is, if she's inspired by her up-close-and-personal encounter with the Simpson family. Her spokesperson tells Reuters that the author has lent her voice to a guest appearance on a future episode of "The Simpsons" that has the Springfield clan visiting England. "Lord of the Rings" star Sir Ian McKellen will also appear in the episode, according to British newspaper The Sun.
The paper quotes "Simpsons" producer Don Payne as saying the episode sends the family to England to find a woman with whom Grandpa Simpson had a wartime fling. "The Simpsons bump into J.K. Rowling outside a bookshop and they talk all about Harry Potter," Payne tells the Sun. The family then catches McKellen in a stage performance of "Macbeth," but Homer violates theatrical superstition by saying the play's name aloud, causing the actor to be hit by a falling scaffold and struck by lightning. (D'oh!)
Series creator Matt Groening is even trying to get Prime Minister Tony Blair to play himself, the Sun reports. "We're very excited about the episode," Payne tells the paper, "and rest assured every British cliché will be trotted out to get a laugh."